CN116286671A

CN116286671A - Salmonella phage SP8, phage composition and application thereof

Info

Publication number: CN116286671A
Application number: CN202310027301.2A
Authority: CN
Inventors: 张召佐; 孙虎芝; 刘广芹; 王翠
Original assignee: Qingdao Phagepharm Bio Tech Co ltd
Current assignee: Qingdao Phagepharm Bio Tech Co ltd
Priority date: 2023-01-09
Filing date: 2023-01-09
Publication date: 2023-06-23

Abstract

The invention discloses a salmonella phage SP8, a phage composition and application thereof, wherein the phage is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 2022, 8 and 12 days and a preservation number of CGMCC No.45256; the phage SP8 with the virulent broad-spectrum can resist gastric acid, can stay in the intestinal tract for a long time, and can effectively prevent and control salmonella infection of poultry through the intestinal tract. The SP8 phage can be prepared into medicines for preventing and treating salmonellosis, environmental disinfectants, feed and water additives, detection kits and the like for application, and in addition, the phage is high-temperature resistant, acid and alkali resistant, easy to carry out industrial production, can be developed in various dosage forms, and the medicines or disinfectants prepared from the phage also have the advantages of safety, green and environment friendliness.

Description

Salmonella phage SP8, phage composition and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to salmonella phage SP8, phage composition and application thereof.

Background

Among Salmonella are human pathogenic bacteria, veterinary pathogenic bacteria, and human and veterinary pathogenic bacteria. Infection caused by salmonella, the body often presents enteritis (symptoms such as diarrhea and vomiting), acute septicemia, typhoid (gastroenteritis and even hemorrhage) and massive inflammatory cell infiltration, and death can be caused seriously. It is reported that the food poisoning rate caused by salmonella in China is 70% -80%, and the main reason is caused by infection of animal foods with salmonella. Therefore, the effective prevention and control of salmonella infection has important public health significance for the healthy development of the breeding industry and the maintenance of food safety.

The salmonella disease is not seasonal, can be developed all year round, is extremely easy to infect the poultry within 3 weeks of age, has higher morbidity and mortality, has insignificant clinical symptoms of adult poultry disease, has low morbidity, is mostly a recessive carrier, can not completely remove salmonella by organisms, can still exist in specific host cells, does not show significant clinical symptoms, and is carried out in vivo for a long time. The vertical transmission characteristic of salmonella pullorum also benefits from the strong tolerance of salmonella pullorum to various antibacterial components in egg white. Salmonella infection can cause growth retardation of broiler chickens, reduced fertility of laying hens, reduced egg yield and eggshell quality, serious death of chickens, economic loss and even influence on public health safety.

At present, the prevention and control of salmonella infection mainly takes antibacterial drugs, but the use of the antibacterial drugs is unreasonable or the blind abuse leads to the occurrence of drug-resistant salmonella, even serious multi-drug resistance problems occur, the treatment effect is poor, animals die, the cultivation cost is increased, and serious economic loss is brought to the breeding industry. Therefore, the problem of multiple drug resistance of salmonella has become a major challenge for the aquaculture industry and even for humans, and development of a green and novel biological agent is needed to solve the problem.

Phage are widely found in nature, in large numbers, and about 10 on earth ³² And each. The phage can be parasitic in various hosts such as bacteria, various substances of the host bacteria are utilized for growth and propagation, progeny phage is synthesized, and after the progeny phage reaches a certain quantity, the host bacteria are cracked and dead, and a large number of progeny phage are released. Because the phage has high specificity, the phage is used as an antibiotic substitute, the cracking capacity of the phage is not influenced by drug-resistant bacteria, and beneficial bacteria cannot be killed, in addition, the phage is separated from the nature, and the phage belongs to a natural biological agent, and has the advantages of low cost, short screening period and higher safety. Therefore, phages are increasingly used in various fields as a safer antibacterial biological agent.

Although salmonella phage are disclosed at present, the existing salmonella phage has poor tolerance to gastric acid environment (acid environment of stomach and pepsin), has short retention time in intestines and stomach, and influences the effective acting time and effect, so the technology still needs to be further improved.

Disclosure of Invention

Aiming at the problems, the invention provides a novel salmonella phage SP8, a phage composition containing the phage and application thereof, wherein the phage SP8 has strong gastric acid tolerance and can stay in intestinal tracts continuously, has the advantages of safety, green and environment protection, and can be used for preparing medicines, feed additives, environmental disinfectants and kits for treating or preventing salmonella infection diseases of chickens.

In order to solve the problems, the technical scheme of the invention is as follows:

in a first aspect, the present invention provides a salmonella bacteriophage with broad spectrum and strong lytic property, which is isolated from feathers of a chicken farm in Shandong province, and the bacteriophage is named as SP8 and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 2022, 8 months and 12 days, a preservation number of CGMCC No.45256, and a preservation address of: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

The salmonella bacteriophage has a polyhedral head structure and a contractile tail, the head is 53-60nm wide, the head is 60-67nm long, and the tail is about 113-120nm long, and according to the classification method of the International Commission on virus classification (ICTV), the bacteriophage form of the salmonella bacteriophage conforms to the characteristics of the long-tail bacteriophage family, belongs to the long-tail bacteriophage, and is named as vB-SenS-SP8 (SP 8 for short).

The bacteriophage SP8 has strong heat resistance, and still maintains the original activity after 60min of action at 60 ℃; the phage still contained 8.5X10 s by 20min at 80deg.C ⁵ PFU/mL higher potency; the phage has stable activity in pH 5.0-11.0, and has potency of 10 when acting for 3 hr at pH 3.0 ⁷ PFU/mL, 3h potency at pH 12 at 10 ⁴ PFU/mL, which indicates that the phage has a strong stability over a broad pH range.

The phage SP8 is a phage with wide cracking spectrum and strong cracking performance, the total cracking rate of 72 strains of salmonella of different sources stored in a laboratory is 90.28%, for salmonella of different sources, the cracking rate of the phage SP8 on salmonella of chicken origin is 91.43%, and the cracking rate of the phage SP8 on salmonella of 15 strains of pigeon origin is 93.33%; the salmonella lysis rate of the food source is slightly lower.

More importantly, the phage SP8 has stronger tolerance to gastric juice, can be used for a drinking water or feed adding way, keeps higher phage activity after passing through the stomach and intestine, and more effectively blocks salmonella infection by an oral way and reduces the colonization of salmonella in the intestinal tract. The experimental result of tolerance in simulated gastric acid when SP8 is used proves that the titer of the phage after 2 hours of action in artificial gastric juice is maintained at 6.04 multiplied by 108PFU/mL, the phage survival rate is 60.40%, and the survival rate of other phages (SP 4, SPP11, SP12, SP6 and SP 10) after 2 hours of action in artificial gastric juice is basically as low as 0. At the same time, the salmonella phage SP8 resides in the gut for a longer period.

Based on the characteristics, the bacteriophage SP8 is used as a new microorganism with application prospect, and can be used as an antibiotic substitute for pathogen control.

In the present application, phage SP8 includes mutants having a homology of more than 98% or 99% by performing a point mutation or a deletion mutation or an addition mutation while maintaining substantially the same bactericidal activity. Since phages are very prone to mutation during replication, mutants of such phages are also within the scope of the claimed application. The sequence of phage SP8 can be obtained by sequencing the biological material deposited according to the invention by known methods. Screening of phage for mutants that are extremely similar to their trait according to the present invention does not require inventive effort for those skilled in the art.

In a second aspect, the invention also provides a phage composition comprising the aforementioned salmonella phage SP8 and other phages. In practical application, in order to further widen the splitting spectrum of the phage preparation, the difference of splitting spectrums of different phages is fully exerted, advantage complementation is carried out, and the pigeon-derived salmonella phage SP8 and other phages can be combined for use.

Alternatively, the phage composition comprises phage SP8 and one or both of phage SP4 (see, in particular, bulletin number CN 108359644B) and phage SPP11 (see, in particular, bulletin number CN 111254121B). Through the combined use of phage, the cracking spectrum is widened, and different types of salmonella in the chicken farm environment are killed, so that the salmonella can be better prevented and treated.

Wherein, phage SPP11 is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms, and has the following addresses: no. 3 of North Chen West Lu No.1 of the Korean region of Beijing city, the preservation date is 11 month 04 of 2019, and the preservation number is CGMCC No.18868. See patent publication number CN111254121B for specific information.

Phage SP4 with collection number of CGMCC No.14332 and collection unit of China general microbiological culture Collection center with collection date of 2017, 7 and 27, and specific information is shown in patent with publication number of CN 108359644B.

In addition, the phage SP8 can be matched with other phages of different types (inhibiting different pathogenic bacteria which cause the same kind of diseases) for preventing and treating the same kind of diseases, such as acute septicemia infected by different pathogenic bacteria in a mixed mode.

In a third aspect, the invention also provides application of the salmonella phage SP8 or phage composition in preparing medicines, feed additives, environmental disinfectants and detection kits for preventing and treating diseases caused by salmonella infection. The control includes prevention and treatment. The term "preventing" is meant herein to include all actions that inhibit or delay the disease by administering the composition. The term "treatment" is meant herein to include all actions that result in improvement or improvement of the disease by administration of the composition.

Preferably, the salmonella-infected disease comprises chicken salmonellosis, the salmonella comprising chicken-derived salmonella; preferably, the salmonella gallinarum is selected from salmonella pullorum, salmonella typhi, and salmonella enteritidis.

In a fourth aspect, the present invention also provides a phage pharmaceutical preparation, the active ingredients of which comprise the above salmonella phage SP8; preferably, the phage pharmaceutical formulation further comprises phage of other specific pathogenic bacteria.

Preferably, the phage pharmaceutical formulation further comprises other bacteriostatic or bactericidal active ingredients; the pharmaceutical preparation is in the form of oral administration dosage form, external application dosage form or parenteral administration dosage form.

Alternatively, the phage pharmaceutical formulation described above may be administered to the pharmaceutical composition of the present invention by a topical, oral or parenteral route. In particular, the compositions of the present invention may be administered via the percutaneous, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, intranasal and inhalation routes. Preferably, the pharmaceutical formulation is in the form of infusion.

Optionally, the phage pharmaceutical formulation 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 administered active component. In order to formulate the pharmaceutical composition into a liquid formulation, the pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, ringer's solution, buffered saline, albumin infusion, dextrose solution, maltodextrin solution, glycerol and ethanol. They may be used alone or in any combination thereof. Other conventional additives, such as antioxidants, buffers, bacteriostats, and the like, may be added if desired. When also combined with diluents, dispersants, surfactants, binders and/or lubricants, the compositions of the present invention can also be prepared as injections (e.g., aqueous solutions, suspensions and emulsions), or as pills, capsules, granules or tablets.

In a fifth aspect, the application also provides a chicken feed additive or drinking water additive, which comprises the salmonella phage SP8, and the chicken feed additive or drinking water is mixed, so that pigeon house drinking water and feed are disinfected and sterilized, the spread of salmonellosis is avoided from the source, and the prevention or treatment of chicken-origin salmonellosis is effectively achieved.

Optionally, the drinking water additive or feed additive also contains other active ingredients for inhibition or elimination of bacteria in the water; the drinking water additive and the feed additive are in the form of liquid dosage form, powder dosage form or solid dosage form, but are not limited to the three dosage forms.

In a sixth aspect, the present application also provides an environmental disinfectant, the active ingredients of which include salmonella phage SP8 as described above, which can be applied to the disinfection of feed, water and cultivation environments.

In a seventh aspect, the present application further provides the use of the above-described environmental disinfectant in the disinfection of chicken farm environments, in particular, the environmental disinfectant can disinfect breeding environments, including tanks, floors, walls, faeces and litter, by spraying, dipping, or salmonella. Such liquid soaking, spraying forms include, but are not limited to, detergents, disinfectants, decontaminating agents, and the like.

In an eighth aspect, the present application also provides a detection kit comprising phage SP8 as described above. Experiments show that the phage SP8 has cleavage specificity to host bacteria, and the phage SP8 can be applied to rapid detection of salmonella, including but not limited to detection of salmonella in the forms of test paper, test paper boxes and the like, or screening of target pathogenic bacteria in clinical samples, so that the detection sensitivity is effectively ensured.

The invention has the following beneficial effects:

1. the invention provides a strong salmonella phage SP8 with a wide lysis spectrum, which can effectively prevent and treat poultry salmonellosis, and particularly can reduce the salmonella infection rate of chicken groups. The phage has high potency, good temperature stability and strong stability in a wider PH range; meanwhile, the salmonella has the characteristics of strong cracking capacity and wide cracking spectrum; in addition, the salmonella phage can resist gastric acid, stay in the intestinal tract for a long time, and have good prevention and treatment effects on salmonella infection in the intestinal tract.

Based on the characteristics, the phage SP8 can be prepared into medicines, environment disinfectants, feed and water additives, detection kits and the like for preventing and treating salmonellosis, and can effectively prevent and treat the spread of the salmonellosis, and has the effects of reducing the mortality rate of chicken groups, reducing the positive rate of antibodies against the salmonellosis, improving the survival rate of chicken farms and the like; the phage product can be used for a long time to achieve the effect of purifying salmonella in chicken farm.

2. The phage SP8 is obtained from the nature, is easy to carry out industrial production, can be used for developing various dosage forms, and has the advantages of being environment-friendly, safe to use and capable of effectively avoiding the generation of multi-drug-resistant salmonella.

Drawings

FIG. 1 is a plaque photograph of Salmonella phage SP8;

FIG. 2 is an electron microscope picture of phage SP8;

FIG. 3 is the results of a thermostability test for phage SP8;

FIG. 4 shows the pH stability test results of phage SP8;

FIG. 5 is a one-step growth curve of phage SP8;

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention. In the present invention, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

EXAMPLE 1 isolation and purification of Salmonella phages

1. The experimental method comprises the following steps:

taking a proper amount of chicken feather and other samples from a certain farm in Shandong, adding the samples into a 250mL conical flask containing a proper amount of broth, fully and uniformly mixing, and incubating for 2 hours at room temperature; after incubation, centrifuging at 11000rpm for 5min, and filtering for later use; taking 1mL of fresh and proliferated salmonella bacteria liquid, 50mL of the filtrate and 500mL of double concentrated NB broth, uniformly mixing in a 250mL conical flask, and culturing for 16-18 h at 37 ℃ and 170 rpm; centrifuging the culture solution at 11000rpm for 5min; filtering the supernatant with a 0.22 μm microporous filter to obtain phage multiplication liquid. Mixing 200 μL of fresh and proliferated bacterial liquid with 200 μL of the filtrate in a 1.5mLEP tube, and incubating in a constant temperature incubator at 37 ℃ for 5min; then 200 mu L of the mixed solution is added into 5mL of the upper layer culture medium, and the mixture is poured into a culture dish containing the lower layer culture medium after being evenly mixed; after solidification, the cells are inverted and placed in a constant temperature incubator at 37 ℃ for 4 to 6 hours, and plaque formation on the double-layer flat plate is observed.

Digging single plaque on the double-layer flat plate, putting the plaque into a centrifuge tube containing 1mL of broth, putting the centrifuge tube into a 37 ℃ 170rpm for leaching for 30min, and centrifuging for 5min at 11000 rpm; diluting the centrifugated leaching solution to a proper gradient according to a ratio of 10 times, taking 200 mu L of diluent with a proper gradient and 200 mu L of salmonella bacteria solution, uniformly mixing, placing the mixture at 37 ℃ for incubation for 5min, taking 200 mu L of mixed solution, adding the mixed solution into a melted upper layer culture medium, rubbing and uniformly mixing, and pouring the mixture into a flat plate containing a common lower layer culture medium; standing for solidification, and then inverting the mixture to be cultured in a constant temperature incubator at 37 ℃ for 4 to 6 hours; the above procedure was repeated until all plaques were of uniform size.

Adding 200 mu L of salmonella phage leaching solution and 200 mu L of host bacterial solution into 5ml of NB broth culture medium, culturing for 6-8 h in a constant temperature oscillator at 37 ℃ and 170rpm until the culture solution becomes clear and has obvious bacterial fragments, centrifuging for 10min at 11000rpm, and filtering the supernatant with a 0.22 mu m filter to obtain phage multiplication solution; 100 mu L of phage multiplication liquid is diluted to a proper gradient according to a ratio of 10 times, and the salmonella phage titer is detected by adopting a double-layer flat plate method.

2. Experimental results and analysis

As shown in FIG. 1, salmonella phage formed clear plaques on double-layered agar medium plates, no halos around, clearly visible edges, and approximately 2mm in diameter; the titer of the salmonella phage was determined to be 6.8X10 ¹⁰ PFU/mL。

EXAMPLE 2 morphological observations of Salmonella phages

1. The experimental method comprises the following steps:

20. Mu.L of phage proliferation solution was added dropwise to the copper mesh, and the solution was allowed to precipitate for 15min, and the excess solution was removed. 2% phosphotungstic acid (PTA) 15 mu L is added dropwise on a copper mesh, after 5min of dyeing, the excess dye liquor is sucked away, and after drying, the dye liquor is observed by a transmission electron microscope and photographed.

2. Experimental results and analysis

As can be seen in FIG. 2, the head of the salmonella phage is polyhedral, the head is 53-60nm wide and 60-67nm long, and has a non-contractile tail, the tail is about 113-120nm long; according to the classification method of the International Commission on viral Classification (ICTV), the phage morphology of the present application meets the characteristics of the family of long-tailed phages, belonging to the long-tailed phages, and is named vB-SenS-SP8 (SP 8 for short).

EXAMPLE 3 Whole genome analysis of phage SP8

The genome of phage SP8 was extracted by a conventional method, and subjected to whole genome sequencing and sequence analysis, with the following results:

(1) The genome of SP8 is full-length 43823kb, the G+C content is 49.53%, and the base C, G, A, T content is 24.73%, 24.80%, 25.15% and 25.32% in this order. The on-line annotation of the whole genome RAST shows that the genome contains 69 Open Reading Frames (ORFs), the average OFR length is 635bp, of which 23 ORFs function well, and 46 ORFs are hypothetical protein expression sequences. Of these 69 Open Reading Frames (ORFs), 20 were found for structural proteins, mainly including phage structure and packaging proteins (head protein, tail fiber protein, neck protein, capsid protein, and terminal enzyme large subunit, etc.), phage cleavage-related proteins (lytic enzymes), DNA replication and modification-related proteins (restriction endonucleases, DNA binding proteins, intron-containing DNA polymerase precursors, HNH endonucleases, DEAD/DEAH cassette helicases, etc.), other functional proteins (repeated infection immunity proteins). Meanwhile, among 69 ORFs, 64 were ATG at the start codon, GTG at the start codon 2, TTG at the start codon 2, and GGA at the start codon 1. The genome did not contain tRNA gene as analyzed by software tRNAscan-SE. The genome was analyzed by an online tool CGE server to be free of drug resistance genes and virulence genes. The genome is free of lysogenic related genes as analyzed by PHASER.

(2) In the genome of phage SP 8: the tail fiber (tail fibers protein) protein gene sequence related to phage host recognition is shown in sequence 1 in the sequence table, and the amino acid sequence is shown in sequence 2; the sequence of the highly conserved terminal enzyme large subunit (terminase large subunit) protein gene is shown as a sequence 3 in a sequence table, and the amino acid sequence is shown as a sequence 4; the sequence of a DNA polymerase (DNA polymerase) gene is shown as a sequence 5 in a sequence table, and the amino acid sequence is shown as a sequence 6; the gene sequence of the lyase (lysozyme) related to the cleavage capacity is shown as a sequence 7 in a sequence table, and the amino acid sequence is shown as a sequence 8. The relevant information is specifically shown in table 1 below.

TABLE 1 Gene sequence information Table

Example 4 temperature stability of Salmonella phage SP8

1. The experimental method comprises the following steps:

will be 8.0X10 ¹⁰ PFU/mL salmonella phage SP8 proliferation liquid is respectively subjected to three parallel samples at 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ and each temperature, heat preservation is carried out for 20min, 40min and 60min respectively, the samples are immediately cooled in an ice bath after the action is finished, and then phage titers at different temperatures are detected by adopting a double-layer flat plate method. And drawing a SP8 phage heat stability curve by taking the temperature as an abscissa and taking the logarithmic value of phage titer as an ordinate.

2. Experimental results and analysis

As can be seen from the results of FIG. 3, phage SP8 remained active after 60min of action at a temperature within 40℃to 60 ℃; the phage was kept at a titer of 8.5X10 after 20min at 80 ℃ ⁵ PFU/mL; the phage SP8 still maintains a certain activity at 90 ℃ for 20min, and the potency is about 1.20X10% ⁴ PFU/mL. As can be seen, salmonella phage SP8 has a high heat resistance and is compatible with high temperature environments.

Example 5 pH stability of Salmonella phage SP8

1. The experimental method comprises the following steps:

taking three test tubes containing 4.5mL NB broth with different pH values (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13), placing the test tubes containing broth with different pH values into a water bath at 37deg.C for 30min, and adding 0.5mL 8.0X10 respectively after the temperature of broth in the test tubes is stable ¹⁰ PFU/mL phage, shaking thoroughly, mixing, and keeping the temperature at 37deg.CThe bath is used for 1h, 2h and 3h. After the phage is acted for a prescribed time, a proper amount of NaOH or HCl is added into the mixed solution containing phage to adjust the pH to about 7.0, and the action of acid and alkali on phage is stopped. Taking each stop solution, and measuring the change of phage titers of different pH values under different action time by adopting a double-layer flat plate method. And drawing a phage acid-base stability curve graph by taking the lg value of phage titer as an ordinate and taking the pH value as an abscissa.

2. Experimental results and analysis

As shown in FIG. 4, it was found that phage SP8 remained at a potency of 10 in the pH range of 5.0 to 11.0 ¹⁰ PFU/mL, the activity is more stable; at pH 3.0 for 3h, the potency is 10 ⁷ PFU/mL; at pH 1.0 for 1h, the potency is 10 ³ PFU/mL; as can be seen, SP8 is able to withstand certain strong acid environments; at pH 12, the 3h potency was at 10 ⁴ PFU/mL,

pH

13, 1h, potency at 10 ³ PFU/mL; therefore, phage SP8 has strong stability over a wide pH range, and is resistant to a range of strong acid and strong base environments.

Example 6 one-step growth curve of Salmonella phage SP8

1. The experimental method comprises the following steps:

the phage titer and the bacterial concentration were adjusted to a multiplicity of infection of 10, 1mL of each of the phage multiplication solution and the bacterial solution after adjustment was taken and mixed thoroughly (timing was started at this time), incubated at 37℃for 5min, centrifuged at 13000rpm for 30s, the supernatant was discarded, washed 1 time with 5mL of NB broth, and the supernatant was discarded after centrifugation. 5mL of NB broth pre-heated at 37℃was added and the pellet was well suspended and rapidly placed in shaking culture at 170rpm at 37 ℃. From

time

0, 200. Mu.L of each fraction was centrifuged at 11000rpm for 5min, the supernatant was collected, phage titers at different time points were determined by a double-layer plate method, 3 replicates were performed for each gradient, and the average value was calculated. And drawing a one-step growth curve by taking the infection time as an abscissa and the titer of phage in an infection system as an ordinate to obtain the incubation period and the outbreak period of phage SP8, and calculating the outbreak amount.

Burst amount = total number of phage at end of burst/total number of bacteria at beginning of burst

2. Experimental results and analysis

The growth curve of phage SP8 is shown in FIG. 5, and the titer is basically unchanged within 15min after the phage infects host bacteria, and the titer is stabilized at 10 ⁶ PFU/mL shows that the incubation period of phage SP8 is about 15min, the number of phage is increased sharply within 15-60 min after the phage infects host bacteria, the potency increases steadily after 60min, and the potency can reach 10 at this time ¹⁰ PFU/mL, phage SP8 burst period was about 40min, burst size 112 PFU/cell.

Example 7 determination of the optimal multiplicity of infection (MOI) of phage SP8 on Salmonella

1. Experimental method

Phage titer and host bacteria concentration are respectively added into a test tube containing 5mL of NB broth according to the equal proportion of 1:1, 0.1:1, 0.01:1, 0.001:1, 0.0001:1, 0.00001:1 and 0.000001:1, and shake culture is carried out at 37 ℃ at 170rpm for 8-12 h until the liquid becomes clear from turbidity; then, centrifugation is carried out at 11000rpm for 5min, the titers of samples with different proportions are measured by adopting a double-layer plate method, three parallel samples are made for each group, the number of plaques in the plates is counted, and the titers are calculated. The group of MOI with highest phage titers is the optimal multiplicity of infection.

2. Experimental results and analysis

The results of the complex infection assay of phage SP8 are shown in Table 1 below, and the highest titer of phage SP8 proliferation when MOI=0.00001 was 1.10X10 ¹¹ PFU/mL, i.e., optimal MOI, is 0.00001.

TABLE 2 phage SP8 MOI assay results

MOI	SP8/(PFU/mL)	Host bacterium/(CFU/mL)	potency/(PF)U/mL)
				1	1.35×10 ⁹	1.35×10 ⁹	1.20×10 ⁹
0.1	1.35×10 ⁸	1.35×10 ⁹	5.60×10 ⁹
				0.01	1.35×10 ⁷	1.35×10 ⁹	1.20×10 ¹⁰
0.001	1.35×10 ⁶	1.35×10 ⁹	3.60×10 ¹⁰
				0.000 1	1.35×10 ⁵	1.35×10 ⁹	6.00×10 ¹⁰
0.000 01	1.35×10 ⁴	1.35×10 ⁹	1.10×10 ¹¹
				0.0000 01	1.35×10 ³	1.35×10 ⁹	7.50×10 ¹⁰
0.0000 001	1.35×10 ²	1.35×10 ⁹	2.00×10 ¹⁰

Example 8 determination of phage lysis spectra

1. Bacterial strain source: 72 salmonella preserved in laboratory

(1) Classification by source type: salmonella gallinarum 35, salmonella duck 18, salmonella pigeon 15, and Salmonella food 4;

(2) Classification by pathogen type: salmonella pullorum 42, salmonella typhimurium 19, salmonella paratyphi 11.

2. Experimental method

And (3) taking 72 strains of salmonella stored in the experiment, resuscitating in an SS (SS) plate, and then picking single colony for proliferation culture to obtain fresh bacterial liquid. Fresh phage multiplication liquid SP8 and 72 salmonella bacterial liquids are prepared, and a double-layer flat plate method is adopted to detect the splitting spectrum of phage. Meanwhile, the splitting spectrum of 72 salmonella was determined by using the phage SPP11 disclosed in the SP4 and the patent CN111254121A of the patent CN108359644B which have been issued by the present company, and the splitting performance of three different salmonella phages was compared.

The cleavage spectrum was measured using 20 Salmonella strains each as described in patent CN108359644B and patent CN111254121A, and the cleavage efficiencies of SP8, SP4 and SPP11 were compared.

3. Experimental results and analysis

3.1 Comparative analysis of lytic effect of 3 kinds of bacteriophage on 72 strains of salmonella of different sources

(1) Salmonella phage SP8 lyses 65 from 72 different sources of Salmonella stored in the laboratory with a total lysis rate of 90.28%. The phage SP8 has the characteristics of wide cleavage spectrum and strong cleavage performance. Specifically, the phage SP8 can lyse 32 strains of 35 strains of chicken-origin salmonella, and the lysis rate is 91.43%; for 18 duck source salmonella, 16 strains can be cracked, and the cracking rate is 88.89%; 14 strains of 15 strains of pigeon-origin salmonella can be cracked, and the cracking rate is 93.33%; for 4 food strains of salmonella, 3 strains can be lysed, the lysis rate is 75.00%, and the detailed results are shown in Table 3.

(2) Phage SPP11 (patent CN 111254121A) was split against 72 Salmonella strains of different origins at a total split ratio of 72.2%. Specifically, the cleavage rate of the bacteriophage SPP11 on 35 strains of chicken-origin salmonella is 80.0%; the cracking rate of 18 strains of duck-origin salmonella is 72.2 percent; the cracking rate of 15 strains of pigeon-derived salmonella is 60.0%; the cleavage efficiency of Salmonella was 50.0% for 4 strains of food, and the detailed results are shown in Table 3.

(3) Phage SP4 (patent CN 108359644B) was split into 37 strains of 72 strains of Salmonella of different origins, with a total split of 51.39%. Specifically, the cleavage rate of phage SP4 on 35 strains of chicken-origin salmonella is 60.0%; the cracking rate of 18 strains of duck-origin salmonella is 33.3 percent; the cracking rate of 15 strains of pigeon-derived salmonella is 53.3%; the cleavage efficiency of Salmonella was 50.0% for 4 strains of food, and the detailed results are shown in Table 3.

3.2 Comparative analysis of lytic effect of 3 kinds of bacteriophage on 72 strains of salmonella of different pathogen types

(1) Salmonella phage SP8 lyses 20 strains for 24 strains of Salmonella pullorum, 15 strains for 16 strains of Salmonella typhimurium, 8 strains for 11 strains of Salmonella paratyphi, 8 strains for 9 strains of Salmonella enteritidis, and 9 strains for 9 strains of Salmonella typhimurium. The cracking rates were 91.67%, 93.75%, 72.7%,88.88%,100%, respectively, as shown in Table 3;

(2) Salmonella phage SPP11 has a splitting ratio of 83.33%, 56.25%, 63.63%, 88.88% and 77.78% for 24 strains of Salmonella pullorum, 16 strains of Salmonella typhimurium, 11 strains of Salmonella paratyphi, 9 strains of Salmonella enteritidis and 9 strains of Salmonella typhimurium respectively; salmonella phage SP4 has a cleavage rate of 45.83%, 68.75%, 81.82%, 33.33% and 11.11% for 24 Salmonella pullorum, 16 Salmonella avium, 11 Salmonella paratyphi, 9 Salmonella enteritidis and 9 Salmonella typhimurium, respectively, as shown in Table 3;

3.3 Comparative analysis of lytic effect of 3 kinds of bacteriophage on 40 strains of salmonella

The 20 salmonella strains in the patent CN111254121B are randomly selected for carrying out the analysis of the cracking spectrum, and the result is that the SP8 can crack 18 strains, the SPP11 can crack 17 strains, the SP4 can crack 15 strains, and the cracking rates are respectively 90.00%, 85.00% and 75.00%. The specific results are shown in Table 4.

The 20 salmonella strains in the patent CN108359644B are randomly selected for carrying out the analysis of the cracking spectrum, and the result is that 17 strains can be cracked by SP8, 13 strains can be cracked by SPP11, 15 strains can be cracked by SP4, and the cracking rates are 85.00%, 65.00% and 75.00% respectively. The specific results are shown in Table 4.

In conclusion, the salmonella phage SP8 has obviously higher salmonella lysis rate of 72 strains of different sources and different pathogen types adopted in the application than phage SP4 and phage SPP11; and for salmonella selected in patent CN108359644B and patent CN111254121a, the lysis rate of phage SP8 is higher than phage SP4 and phage SPP11.

The results show that the phage SP8 has the advantages of wider cracking spectrum and higher cracking performance, and can effectively prevent and control the infection of salmonella from different sources.

Table 33 cleavage Profile of phages against Salmonella 72 strain

/>

Table 43 lytic spectra of phages against 40 Salmonella strains

Example 9 tolerance test of phage SP8 in simulated gastric acid

1. Experimental method

(1) Preparing artificial gastric juice: taking 16.4ml of dilute hydrochloric acid, adding 800ml of deionized water, fully stirring, adjusting the pH value to 2.5, fixing the volume of the deionized water to 1L, sterilizing at 121 ℃ for 15min under high pressure, cooling to room temperature, adding 10g of pepsin, and mixing to fully dissolve the pepsin.

(2) Phage SP8, SP4 (see CN 108359644A) and SPP11 (see CN 111254121A), which are both chicken-derived Salmonella phages, and 1mL of each of SP6, SP10 and SP12 were taken and added to 150mL Erlenmeyer flasks, respectively, followed by 20mL of the aforementioned artificial gastric juice. Among them, SP6, SP10 and SP12 are chicken-derived salmonella phages which were isolated and stored by themselves in the laboratory, and in the present experiment, comparative analysis was performed only as a control.

The conical flask was placed in a constant temperature incubator at 37℃for 2h at 90r/min, 1mL of the mixture was sampled per hour, diluted to a suitable gradient in 9mL of buffer solution, phage titer was determined by double-layer plate method, and survival rate was calculated.

Survival = (number of phages after fixed time of artificial gastric juice treatment/number of phages at 0h of artificial gastric juice treatment) ×100%

2. Experimental results and analysis

As can be seen from the results in Table 5, phage SP8 was allowed to act in artificial gastric juice for 2 hours at a titer of 1X 10 ⁹ PFU/mL was reduced to 6.04×10 ⁸ PFU/mL; SP4 was allowed to act for 2h in artificial gastric juice, decreasing the potency from 1X 109PFU/mL to 1.09X 105PFU/mL, and still retaining 60.40% survival.

While phage SPP11 was allowed to act in artificial gastric juice for 2h at a titer of 1X 10 ⁹ PFU/mL was reduced to 5.7X10 ² PFU/mL; SP12 in humansThe medicinal liquid has a potency of 1×10 and acts for 2 hr ⁹ PFU/mL was reduced to 8.47×10 ¹ PFU/mL; SP6 was allowed to act in artificial gastric juice for 2h at a potency of 1X 10 ⁹ PFU/mL was reduced to 2.06X10 ⁴ PFU/mL; SP10 was allowed to act in artificial gastric juice for 2h at a potency of 1X 10 ⁹ PFU/mL was reduced to 4.96×10 ³ PFU/mL. As can be seen from the comparison of the phages in the above experimental results, phage SP8 was more resistant to simulated gastric fluid.

TABLE 5 potency Change of phages in Artificial gastric juice at various times (PFU/mL)

Example 10 metabolism experiment of phage SP8 in the intestinal tract of chickens

1. Experimental method

SPF chicken of 3 days old orally taken 1X 10 ¹⁰ 5 chickens were randomly dissected through PFU salmonella phage SP8 at 0.5h, 2h, 6h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, 168h and 192h, the duodenum, jejunum, ileum, cecum and rectum were taken and weighed, and the phage content in the different intestinal segments was detected by a double-layer plate method, and the average content was calculated.

2. Experimental results and analysis

Salmonella phage SP8 was detected in the twelve rectum for 0.5h at 5.19X10 ⁸ PFU/g, phage empty at 120 h; the metabolism of phage in jejunum and ileum is more consistent, and the phage content reaches peak value in 6-12 h; the cecum and intrarectal phages were found to be the longest, with higher levels of cecum phages at 144h, 5.43X10 ⁶ PFU/g, small amounts of phage were still detectable in cecum and rectum at 192 h. From the above data, salmonella phage SP8 was able to stay in chicken intestine for a long period of time.

TABLE 6 phage metabolism results in different intestinal segments (PFU/g)

Example 11 action of phage SP8 on Salmonella in the intestinal tract of chickens

1. Experimental method

Randomly dividing 20 SPF chickens into 2 groups, each group of 10 chickens being control group and phage group respectively, wherein the phage dosage is 1×10 ⁸ pfu/chicken. All chickens drink water freely to eat, and after taking phage, the oral poison eliminating agent is 1X 10 after 2 hours ⁷ Salmonella enteritidis of cfu. The control group was filled with 0.5mL of physiological saline. 1 day, 3 days, and 7 days after the detoxification, randomly taking 5 chickens from each group with CO ₂ Killing by asphyxia, dissecting, taking out the cecum content, adding bacteria in magnesium chloride malachite green bacteria-adding liquid after adding bacteria by using broth, inoculating the bacteria-adding liquid on a xylolysin deoxycholate culture medium (XLD), culturing for 24-36 h, and observing whether black salmonella colonies grow.

2. Experimental results and analysis

The control group 10 chickens are positive in terms of salmonella enteritidis detection in cecum on

days

1, 3 and 7, the positive rate of salmonella enteritidis detection in phage group on the first day is 40%, and the detection is not negative on

days

3 and 7. Demonstrating that salmonella enteritidis was cleared from cecum on day 3 after oral administration of salmonella phage SP8.

TABLE 7 Salmonella enteritidis detection results

Example 12 safety test of phage SP8

1. Experimental method

30 SPF chicks with 1 day age are selected and divided into a phage group and a control group, and the phage group is orally taken for 1 multiplied by 10 ¹⁰ PFU phage 0.2mL, and feeding and observing for 7 days. Control group oral equivalent dose does not existPhysiological saline. And (3) performing a section examination to observe pathological changes of heart, liver, spleen, lung, kidney, brain, intestinal tract, and observing states such as spirit, ingestion and the like in the feeding process.

2. Experimental results and analysis

No disease or toxic symptoms, mental states and normal feeding were observed in phage and control groups throughout the dosing period. Through detailed clinical section observation, the main organs and intestinal tracts of animals are normal whether in a phage group or a control group. The phage SP8 has higher safety and has no adverse effect on animal organisms.

Example 13 phage SP8 Sterilization test against chicken farm Environment

1. Experimental method

Randomly selecting 2 chicken houses of 3 days old in a chicken farm in the Shandong province, and detecting the salmonella content in the environment of each chicken house before disinfection; the test group was sterilized with phage preparation, phage disinfectant content 10 ⁶ PFU/mL, spray volume of 10mL/m ² The method comprises the steps of carrying out a first treatment on the surface of the The control group was sterilized with peracetic acid (1:500) disinfectant. And detecting the salmonella content in the environment again after the two groups are disinfected for 30 min.

The detection method comprises the following steps: taking 5 points in the center point and the 4 points at the corner of the house as test points, placing 2 XLD agar plates at each point for sampling, placing the culture dishes in a constant temperature incubator at 37 ℃ after sampling, and recording the bacterial colony numbers in the culture dishes after culturing for 18-24 hours.

Counting the total number of colonies according to the Orthosiphon formula, wherein C is the total number of colonies per cubic meter (CFU/m) ³ ) The method comprises the steps of carrying out a first treatment on the surface of the N is the colony number of each dish; area of Petri dish (cm) ² ) The method comprises the steps of carrying out a first treatment on the surface of the T is sampling time (min).

2. Experimental results and analysis

As shown in Table 8, after the disinfection by phage SP8, the salmonella elimination rate in the chicken house is 87.93%; after being disinfected by an oxyacetic acid disinfectant, the salmonella elimination rate in the henhouse is 51.92%; the phage SP8 has obviously better effect of killing salmonella in henhouse environment than peroxyacetic acid, and can be used as a novel biological environment disinfectant for popularization and application.

TABLE 8 Salmonella colony counts before and after phage SP8 and peracetic acid disinfectant sterilization

EXAMPLE 14 lytic assay of phage SP8 against non-host bacteria

1. Experimental method

10 strains of colibacillus, 5 strains of Proteus, 5 strains of staphylococcus and 5 strains of clostridium welicum are selected, 25 different types of non-host bacteria are selected, and phage SP8 lysis spectrum measurement experiments are carried out according to the measurement method of the lysis spectrum in the previous embodiment.

2. Experimental results and analysis

No transparent plaques were found in the double-layer plates of phage SP8 lysis spectrum and 25 non-host bacteria, indicating that phage SP8 could not recognize these non-host bacteria, indicating that the test phage SP8 has very strong host specificity and no damage to the microbial community, which can be used to prepare detection kits.

It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present teachings and concepts, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the accompanying claims.

Claims

1. The salmonella phage SP8 is characterized in that the preservation number is CGMCC No.45256.

2. A phage composition comprising salmonella phage SP8 of claim 1 and other phages.

3. The phage composition of claim 2, wherein the other phage comprises: one or two of phage SP4 and phage SPP11; wherein, the deposit number of phage SP4 is: CGMCC No.14332; the preservation number of phage SPP11 is CGMCC No.18868.

4. Use of a salmonella bacteriophage SP8 of claim 1 or a bacteriophage composition of claim 2 or 3 for the manufacture of a medicament, feed additive, environmental disinfectant, food preservative and detection kit for the prevention and treatment of a disease caused by salmonella infection.

5. A phage pharmaceutical preparation comprising as an active ingredient the salmonella phage SP8 of claim 1 or the phage composition of claim 2 or 3; preferably, the phage pharmaceutical formulation further comprises phage of other specific pathogenic bacteria.

6. The phage pharmaceutical formulation of claim 5, wherein the pharmaceutical formulation is in the form of an oral administration dosage form, a topical dosage form, or a parenteral administration dosage form.

7. A chicken feed additive or drinking water additive comprising a salmonella phage SP8 of claim 1 or a phage composition of claim 2 or 3.

8. An environmental disinfectant, characterized in that the active ingredient comprises the salmonella phage SP8 of claim 1 or the phage composition of claim 2 or 3; preferably, other active ingredients for the inhibition or elimination of bacteria in the environment are also included.

9. Use of an environmental disinfectant according to claim 8 for the disinfection of chicken farm environments, including tanks, floors, walls, faeces and litter, by spraying, dipping to disinfect the cultivation environment, the feeding implements, salmonella.

10. A test kit comprising the salmonella phage SP8 of claim 1.