CN-121975745-A - Preparation and application of cross-host multivalent phage and bacteriostat comprising phage

Abstract

The invention relates to phage, in particular to preparation and application of a cross-host multivalent phage and a bacteriostat comprising phage, wherein the multivalent phage is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2026045. The invention discovers salmonella phage CCTCC NO: M2026045 which is multivalent phage for the first time, can cross-host infection and crack various important enteropathogenic bacteria of livestock and poultry such as salmonella, escherichia coli, shigella, klebsiella pneumoniae, proteus mirabilis and the like, solves the problem of narrow host range of single phage, and improves the prevention and control efficiency. The microbial ecological balance of the intestinal canal can be regulated by inhibiting the overgrowth of the gram-negative pathogenic bacteria, so that the aim of effective prevention and control is achieved.

Inventors

• ZHANG HUI
• BAO HONGDUO
• WANG RAN

Assignees

• 江苏省农业科学院

Dates

Publication Date

20260505

Application Date

20260127

Claims (10)

1. 1. The salmonella phage is characterized in that the salmonella phage is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2026045 and is classified and named as salmonella phage vB_ SaeS _S33 (Salmonella phage vB _ SaeS _S33).
2. 2. A method of culturing the salmonella phage of claim 1, comprising the steps of: 1) Centrifuging the salmonella enteritidis S33 culture, and collecting precipitate; 2) And (3) re-suspending the precipitate in the step (1) by utilizing an LB culture medium to obtain a bacterial suspension, adding the salmonella phage CCTCC NO: M2026045 into the bacterial suspension, culturing for 6-8 h at 37 ℃, centrifuging, and taking a supernatant to obtain phage lysate.
3. 3. The method of claim 2, wherein the salmonella culture concentration in step 1) is 1 x 10 8 cfu/mL~1×10 9 cfu/mL.
4. 4. A bacteriostat comprising the salmonella phage of claim 1 or phage lysate obtained by the method of claim 2.
5. 5. The bacteriostat of claim 4, further comprising a pharmaceutically and animal-raising acceptable excipient.
6. 6. The bacteriostat of claim 5, wherein the pharmaceutically acceptable excipient comprises one or more of a buffer, a metal ion, and a surfactant.
7. 7. The bacteriostat of claim 5, wherein the bacteriostat is in the form of a liquid, a lyophilized powder, a spray or a premix.
8. 8. The salmonella bacteriophage of claim 1 or the application of the bacteriostat of any one of claims 5-7 in preparing a medicament for inhibiting and/or eliminating salmonella of pathogens, wherein the pathogens are any one or more of salmonella, escherichia coli, shigella, klebsiella pneumoniae and proteus mirabilis.
9. 9. The salmonella bacteriophage of claim 1 or the use of the bacteriostat of any one of claims 5-7 in the preparation of a medicament for preventing and treating an intestinal disease of an animal.
10. 10. The use according to claim 9, wherein the animal comprises a chicken, duck or pig.

Description

Preparation and application of cross-host multivalent phage and bacteriostat comprising phage Technical Field The invention relates to phage, in particular to preparation and application of a cross-host multivalent phage and a bacteriostat comprising phage. Background In livestock and poultry farming, intestinal diseases caused by mixed infection of salmonella (Salmonella spp.), escherichia coli (ESCHERICHIA COLI), shigella spp.), klebsiella pneumoniae (Klebsiella pneumoniae), proteus mirabilis (Proteus mirabilis) and the like are important problems affecting animal health and causing economic losses. At present, prevention and control of these bacterial infections mainly depend on antibiotics, but long-term abuse of antibiotics has led to increasingly serious bacterial resistance, and the problem of drug residues threatens food safety and public health. Therefore, there is an urgent need to develop a safe, efficient and residue-free antibiotic substitute. The phage is used as a virus capable of specifically infecting and lysing bacteria, has the core advantages of strong host specificity, self proliferation, high biosafety, no obvious toxic or side effect and the like, and is a novel biological prevention and control agent with great application potential in the fields of biological medicine and biological prevention and control. However, the extremely strong host specificity can give the capability of precisely targeting and lysing target bacteria, and simultaneously greatly limits the practical application range of the target bacteria in complex bacteria conditions, thereby becoming a key bottleneck for restricting the large-scale application of the target bacteria. Existing studies have shown that some phages, especially members of the family long-tailed phages, have the ability to recognize and lyse across bacterial species. The core mechanism is that the identification and the cleavage of the phage to gram-negative bacteria mainly depend on the specific combination between tail fiber protein and bacterial surface O antigen (polysaccharide side chain of lipopolysaccharide LPS). The highly specific interaction enables the phage to efficiently invade and lyse host bacteria having the same O serotype, thereby achieving precise regulation of the target flora. Specifically, part of phage is firstly adsorbed by fiber protein with low affinity and non-specificity, and then O antigen polysaccharide is locally degraded by means of glycoside hydrolase or deacetylase carried by the phage, so that lower conserved core polysaccharide or outer membrane protein is exposed. These structures can act as high affinity secondary receptors, helping phage to complete the subsequent invasion process. Because of the homology between O antigens of different bacteria, they provide a common "molecular portal" for phages, enabling a single phage or a specific phage cocktail to lyse multiple pathogenic bacteria. From the molecular structure, the glycosyl composition (such as common monosaccharides such as GlcNAc and Gal) of the O antigen and the core polysaccharide skeleton structure have high conservation among bacteria of different bacteria. Based on this property, the receptor binding proteins of phage (e.g., fiber proteins) can evolve to form specific binding domains for these conserved epitopes, thereby completing the recognition of a variety of bacteria across bacterial species boundaries. The tail fiber protein far-end module responsible for initial adsorption function has structural plasticity, and can adapt a single binding domain to various O antigen chains with similar structures by means of gene recombination or point mutation to form a cross-host recognition effect of 'one key for unlocking multiple locks'. Although the regulation and control of the phage cross-host activity can be realized by manually modifying the fiber protein far-end module, the technical path has extremely high risk of gene pollution, the genetic stability of the modified phage is easy to be out of control, and the phage is difficult to be converted into a practical application scene. In contrast, the phage naturally possessing cross-host lytic activity is easier to guarantee due to no need of artificial modification and biological safety, and shows unique advantages in practical application, but the acquisition process of the phage faces great challenges, and the screening difficulty is extremely high and has remarkable randomness. Specifically, the host spectrum of the natural phage is determined by the gene sequence of the natural phage, the abundance of the trans-host active strain in the natural phage library is extremely low, and the lack of specific screening markers leads to the need of large-scale library screening and repeated verification, which not only consumes time and labor, but also is difficult to accurately predict the screening result, thus greatly limiting the development and application of high-quality phage resources. Disclo