CN117586966B - Acid and alkali resistant clostridium perfringens bacteriophage RDP-CP-22005 and application thereof - Google Patents
Acid and alkali resistant clostridium perfringens bacteriophage RDP-CP-22005 and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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- A61K35/76—Viruses; Subviral particles; Bacteriophages
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10211—Podoviridae
- C12N2795/10232—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
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Abstract
The invention discloses an acid and alkali resistant clostridium perfringens bacteriophage RDP-CP-22005 and application thereof, wherein the bacteriophage is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 45449 in the year 2023 and the month 23. The invention also provides application of the phage or bactericidal composition in preparing at least one of the following products: (1) a product that kills or inhibits clostridium perfringens; (2) A product for preventing and/or treating diseases of poultry caused by clostridium perfringens; (3) A product for preventing and/or treating inflammatory reactions caused by clostridium perfringens. The phage has good acid and alkali resistance under the condition that the pH value is 2.0-13.0, and ensures that the activity of the phage is not lost. The phage has a good cracking effect on clostridium perfringens, can crack hosts of various serotypes, has a wide cracking spectrum, can replace antibiotics and resist corrosion of gastric acid, so that intestinal diseases of the hosts are effectively treated, and meanwhile, the phage is environment-friendly.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to an acid and alkali resistant clostridium perfringens bacteriophage RDP-CP-22005 and application thereof.
Background
Clostridium perfringens (Clostridium perfringens) is an anaerobic bacillus and gram staining is positive. Originally isolated from a highly rotten cadaveric vascular tissue by the U.S. pathologist w.h.welchii et al, one is named by his name and is also known as clostridium welchii. The academic name has been revised many times later due to the change in international bacterial naming regulations and is ultimately more named clostridium perfringens. The length of the bacterial cells is 3-9 μm, the width is 0.6-2.4 μm, the two ends of the bacterial cells are rounded to be thick rod-shaped, and the edges of the bacterial cells are straight. The cell morphology is not fixed, and tends to be spherical at the early stage of cell growth, while tadpoles, filaments and grains are often present at the end of growth due to exhaustion of nutrient components of the medium. The mirror may be arranged singly or in short chains, and sometimes in pairs.
The bacterium is capable of producing a variety of strong exotoxins, twelve of α, β, ε, γ, η, δ, iota, θ, κ, λ, μ, and ν are currently known. Of these, the four exotoxins of alpha, beta, epsilon and iota are all proteins, and have enzymatic activity and antigenicity. They are the most predominant lethal and genotyping toxins. Clostridium perfringens can be divided into, depending on the main exotoxin secreted: A-E5 toxin types, each of which can cause morbidity in animals and humans to varying degrees.
Clinically, the traditional basic approach to the treatment of clostridium perfringens is to use antibiotics. In recent years, with the massive use of antibacterial drugs, particularly in China, antibiotics are added into feed for a long time as feed additives, so that pathogenic bacteria generate drug resistance, the drug resistance spectrum is continuously expanded, the transmission speed is also faster and faster, and the health and public health safety of people are seriously threatened. However, with the advent of antibiotics, the emergence of resistant strains, the treatment of clostridium perfringens is currently very difficult.
Phage, also known as bacterial viruses, are microorganisms that complete their proliferation in bacteria and release themselves into the environment by lysing the bacteria. Compared with antibiotic treatment, phage therapy has the advantages of low metabolic efficiency, low minimum bacterial killing dosage, high specificity, high sterilizing effect, high screening speed and the like.
In recent years, the research on phage has been greatly developed, and the application research range of phage is wide at home and abroad, including the aspects of prevention and treatment of human diseases, prevention and treatment of animal diseases, removal of pathogenic bacteria in food, prevention and treatment of bacterial infection of crops, and lysis of bacterial biofilm. Although phage application is promising, it is not neglected that phages often have a narrow host spectrum, and that one phage usually only lyses one bacterium. In addition, the phage is usually orally taken in the practical production and application process, and gastric acid and other components are easy to inactivate the phage when the phage passes through the digestive tract.
At present, no acid-base resistant phage product capable of preventing and treating clostridium perfringens disease exists, so that a phage product which can resist strong acid and strong alkali, has wide cleavage spectrum, has prevention and treatment functions, has obvious effect and is inherited stably is developed for treating clostridium perfringens disease.
Disclosure of Invention
Aiming at overcoming the defects of the prior art, the invention provides an acid and alkali resistant clostridium perfringens bacteriophage RDP-CP-22005 and application thereof. The acid and alkali resistant clostridium perfringens bacteriophage RDP-CP-22005 provided by the invention can resist strong acid and alkali, ensure that the activity of the bacteriophage is not lost, can replace antibiotics, can resist the corrosion of gastric acid, effectively treat intestinal diseases of a host, and is environment-friendly.
The above object of the present invention is achieved by the following technical scheme:
In a first aspect, the invention provides an acid and alkali resistant clostridium perfringens bacteriophage RDP-CP-22005 isolated from cecum of sick birds in a disease-causing farm in Shandong, deposited with the China general microbiological culture Collection center at 3/23 of 2023 with the following deposit addresses: the preservation number of the Qingyang area North Star Xili No. 1 and 3 of Beijing is CGMCC No.45449.
Observed by electron microscopy: the phage RDP-CP-22005 has a polyhedral, stereospecific head, surrounding nucleic acid, with a diameter of about 46nm, a tail of about 22nm, a tail sheath, and a neck connecting the head and tail. The phage is identified according to the classification standard of the International Commission on viral classification (ICTV), the morphology accords with the characteristics of the order of the tail viruses, and the phage belongs to the family of the short tail viruses.
The phage RDP-CP-22005 was cultured for 8 hours under MOI=0.1, and its titer was as high as 7.93×10 9 PFU/mL.
The phage can still keep self stability after being treated for 2 hours under the condition that the pH value is 2.0-13.0, still has higher activity under the strong acid and alkali environment, still keeps higher lysis effect on host bacteria, and has strong acid and alkali resistance compared with other existing phage; based on the excellent biological characteristics, the phage can adapt to the harsh environment of strong acids and strong bases.
In the process of 30 times of passage of the phage, the titer is always kept high, no obvious fluctuation appears, and the phage can be stably inherited and is suitable for large-scale industrial production.
The bacteriophage has better lysis effect on clostridium perfringens, and experiments prove that the bacteriophage has a lysis rate of 90.9% on clostridium perfringens, can lyse hosts of various serotypes of A type, B type, C type, D type and E type, for example, in the embodiment of the invention, the lysis rate of 82.4% on the selected clostridium perfringens of A type (17 strains can lyse 14 strains), the lysis rate of 100% on the selected clostridium perfringens of B type (6 strains can lyse), the lysis rate of 90.9% on the selected clostridium perfringens of C type (11 strains can lyse 10 strains), the lysis rate of 100% on the selected clostridium perfringens of D type (5 strains can lyse) and the lysis rate of 100% on the selected clostridium perfringens of E type (5 strains). The phage RDP-CP-22005 has excellent cracking performance on clostridium perfringens of various serotypes, has a wide cracking spectrum, has a wide cracking range and a wide application range, and has good application prospect in killing clostridium perfringens in the environment and preventing and treating diseases caused by clostridium perfringens.
In a second aspect, based on the same inventive concept, the present invention also provides a bactericidal composition comprising clostridium perfringens bacteriophage RDP-CP-22005 as described above.
In a third aspect, based on the same inventive concept, the present invention also provides the use of clostridium perfringens bacteriophage RDP-CP-22005 or a bactericidal composition as described above for the preparation of at least one of the following products: (1) a product that kills or inhibits clostridium perfringens; (2) A product for preventing and/or treating diseases of poultry caused by clostridium perfringens; (3) A product for preventing and/or treating inflammatory reactions caused by clostridium perfringens. Preferably, the present invention provides the use of clostridium perfringens bacteriophage RDP-CP-22005 or a bactericidal composition as described above for the preparation of a product for the prevention and/or treatment of diseases in poultry caused by clostridium perfringens. Preferably, the poultry is chicken. The term "preventing" is meant herein to include all actions that inhibit or delay the disease by administering the phage. The term "treatment" is meant herein to include all actions that result in an improvement or improvement of the disease by administration of the phage.
In a fourth aspect, based on the same inventive concept, the present invention also provides a phage pharmaceutical preparation, the active ingredient of which comprises clostridium perfringens phage RDP-CP-22005 as described above.
A phage preparation as described above, further comprising a pharmaceutically acceptable carrier, wherein the pharmaceutical preparation is in the form of an oral administration form or an injection administration form. Preferably, the phage pharmaceutical preparation is in an oral administration form, the phage adopts an oral administration mode in the actual production and application process, and components such as gastric acid and the like are easy to inactivate the phage when the phage passes through the alimentary canal, but the inventor of the application surprisingly discovers that the phage RDP-CP-22005 can resist strong acid and strong alkali, ensures that the activity of the phage cannot be lost, and achieves the effect of preventing or treating clostridium perfringens.
The beneficial effects of the invention are as follows:
1. The acid and alkali resistant clostridium perfringens bacteriophage RDP-CP-22005 provided by the invention has good acid and alkali resistance under the condition that the pH value is 2.0-13.0, can resist strong acid and alkali, and ensures that the activity of the bacteriophage is not lost. The phage can replace antibiotics, resist corrosion of gastric acid, effectively treat intestinal diseases of a host, and is environment-friendly.
2. The acid and alkali resistant clostridium perfringens bacteriophage RDP-CP-22005 provided by the invention has a relatively wide cracking spectrum, has a cracking rate of 90.9% for 44 clostridium perfringens strains from different regions such as Shandong province, jiangsu province, heilongjiang province, hebei province and the like, has a wide application range, and provides a bacteriophage source for industrially producing bacteriophage and preventing and treating diseases caused by clostridium perfringens.
3. The phage provided by the invention is obtained from nature, can be inherited stably, is suitable for large-scale industrial production, is safe to use and is environment-friendly.
Drawings
FIG. 1 is a plaque photograph of phage RDP-CP-22005;
FIG. 2 is an electron micrograph of phage RDP-CP-22005;
FIG. 3 is a one-step growth curve of phage RDP-CP-22005;
FIG. 4 is a graph showing the results of a pH stability test of phage RDP-CP-22005;
FIG. 5 is a graph showing acid and alkali resistance of different phages;
FIG. 6 is a graph showing the results of a temperature sensitivity test for phage RDP-CP-22005;
FIG. 7 is a graph showing the results of a genetic stability test of phage RDP-CP-22005;
FIG. 8 is a graph showing the survival of phage RDP-CP-22005 against broiler chickens.
Detailed Description
The following description of the embodiments of the present invention will clearly and fully describe the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, 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 be 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. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the present invention, clostridium perfringens CP-20032 or simply referred to as "CP-20032", clostridium perfringens bacteriophage RDP-CP-22005 or simply referred to as "bacteriophage RDP-CP-22005" or "RDP-CP-22005".
Example 1 isolation and characterization of Clostridium perfringens CP-20032 as a host
Sampling from a disease-causing farm in Shandong province, performing aseptic operation to obtain diseased poultry cecum, streaking on a selective culture medium, culturing for 18-24h in an anaerobic environment at 37 ℃, wherein bacteria form dome-shaped colonies on a blood agar culture medium, and the colonies are smooth in surface, 1-2.5mm in radius, semitransparent and neat in edge; grass green colonies with double-layer hemolysis rings around are selected on a blood agar basal plate (5% sheep blood plus 1% glucose), inoculated on a pancreatic month indicator-sulfite-cycloserine agar foundation (TSC), placed in an anaerobic environment and cultured for 18-24 hours at 45 ℃; the typical colony is picked and purified for 3-5 times continuously until the colony morphology is consistent, then single colony is picked and inoculated into 5mL of liquid thioglycolate (FTG) broth culture medium, and the culture is carried out for 12 hours in an anaerobic incubator at 37 ℃ to obtain even turbid bacterial suspension. And then determining the bacterial strain as pathogenic clostridium perfringens through 16SrRNA molecular identification and serotype identification, namely, naming one bacterial strain as CP-20032, streaking the identified bacterial strain on a blood agar basal plate culture medium plate, culturing for 12 hours, scraping all bacterial colonies into 60% glycerol-FTG liquid by using an inoculating loop, and storing in a refrigerator at the temperature of minus 80 ℃.
EXAMPLE 2 isolation and identification of phage RDP-CP-22005
(1) And (3) treating the disease materials: taking sick chickens from a farm, dissecting and reserving cecum, taking the cecum content by aseptic operation, adding 2mL of sterilized FTG liquid, fully soaking the cecum content, centrifuging at 10000rpm for 30min, taking supernatant, and filtering the supernatant with a 0.22 mu m filter to reserve;
(2) Preparing phage enrichment liquid: adding 0.2mL of bacterial suspension and 1mL of filtrate into 5mL of FTG broth, culturing at 37 ℃ in an anaerobic box for overnight, centrifuging at 10000rpm for 5min, and filtering the supernatant with a 0.22 mu m filter to obtain filtrate for later use;
(3) Phage isolation: separating phage by double-layer plate method, mixing phage enrichment filtrate 0.1mL and host clostridium perfringens bacterial suspension 0.2mL uniformly, placing in 37 deg.C water bath for 10min, spreading double-layer plates, culturing in anaerobic incubator at 37 deg.C for 6-8h, soaking transparent spot in SM buffer 1mL at 4 deg.C for 12h, collecting leachate 0.1mL and bacterial suspension 0.2mL in 37 deg.C water bath for 10min, spreading double-layer plates, culturing in anaerobic incubator at 37 deg.C for 4-6h, and purifying for 5 times. RDP-CP-22005 formed plaques on double plates with a diameter of 1mm-3mm as shown in FIG. 1.
Phage RDP-CP-22005 preservation method: mixing phage multiplication solution with 60% glycerol at volume ratio of 1:1, and storing in-80deg.C refrigerator or liquid nitrogen.
EXAMPLE 3 Electron microscopic observation of phage RDP-CP-22005
20 Mu L of liquid containing crude phage particles is dripped on a copper mesh, natural precipitation is carried out for 15min, excessive liquid is sucked off from the side face by using filter paper, a drop of 2% phosphotungstic acid (PTA) is added on the copper mesh for dyeing phage for 10min, then the dyeing liquid is sucked off from the side face by using filter paper, and after a sample is dried, the shape of phage is observed by using an electron microscope, as shown in figure 2.
The observation results: phage RDP-CP-22005 has a polyhedral, stereosymmetrical head, surrounding nucleic acid, with a diameter of about 46nm, a tail of about 22nm, a tail sheath, and a neck connecting the head and tail. The phage were identified according to the International Commission on classification of viruses (ICTV) and classified as having the order of the Rheumatoid, brevibacteriaceae.
The phage is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 3 and 23 of 2023, and the preservation address is: the collection number of the Qingyang area North Star Xilu No.1 and 3 of Beijing is CGMCC No.45449, and the Qingyang area North Star Xilu No.1 is classified and named as phage, and the sequence of the phage is shown as SEQ ID No. 1.
EXAMPLE 4 ORF prediction and functional annotation of the Whole genome of phage RDP-CP-22005
Concentrating the high-purity phage solution, and performing full genome second-generation sequencing assembly by adopting a second-generation Illumina Hiseq sequencing technology.
The whole genome sequence of phage RDP-CP-22005 was used to encode gene prediction for the newly sequenced genome using GENEMARKS software. And (3) respectively comparing the protein sequences of the predicted genes with Nr and Swiss-Prot database rows blastp, so as to obtain annotation information of the predicted genes. And performing blastp comparison with eggNOG databases to obtain COG annotation results corresponding to the genes, and performing functional classification on the proteins according to the COG annotation results. The possible virulence genes and drug resistance genes are checked one by one through an online website Virulence Factor Database, VFDB (http:// www.mgc.ac.cn/VFs/main. Htm) and CARD (https:// CARD. Mcmaster. Ca /), and finally the function annotation of the phage is completed.
The phage whole genome assembly results are shown in Table 1.
TABLE 1 statistical Table of phage genome assembly results
Phage genome ORF analysis and functional annotation are as follows, phage gene information statistics are shown in table 2.
TABLE 2 statistical Table of phage Gene information
The amino acid sequence of the phage RDP-CP-22005 genomic ORF protein was compared with the amino acid sequences in the Nr and Swiss databases, and 21 open reading frames were predicted, with an average length of 859bp, and the potential functions of 12 proteins were predicted by functional homology, with the remaining 9 proteins having no similarity to known phage or bacterial proteins. Phage RDP-CP-22005 was categorized into 3 parts by protein function: cell wall/membrane/envelope biogenesis (ORF 11, OR 13), DNA replication and regulation related genes (ORF 4, ORF 21) and phage structure related proteins (ORF 5, ORF9, ORF10, ORF12, ORF15, ORF16, ORF 18). Wherein ORF13 encodes a peptidoglycan hydrolase: N-acetylmuramyl-L-alanine amidase (N-acetylmuramoyl-L-ALANINE AMIDASE) capable of specifically cleaving the amide bond between the lactoyl group of muramic acid and the alpha-amino group of L-alanine. Phage RDP-CP-22005 contains no drug resistance genes and no virulence genes, and contains no lysogenic related genes.
Example 5 determination of optimal multiplicity of infection of phage RDP-CP-22005
1. The experimental method comprises the following steps:
Phage proliferation solution and host were added to FTG broth at a ratio of multiplicity of infection of 100, 10, 1, 0.1, 0.01, 0.001 and the total volume of the culture system was ensured to be the same. After 8h of culture in an anaerobic box at 37 ℃, the culture was centrifuged at 12000r/min for 5min at normal temperature, and the supernatant was spread on double plates to determine the titer. See in particular table 3.
TABLE 3 determination of optimal multiplicity of infection
2. Experimental results and analysis:
As can be seen from Table 3, when the multiplicity of infection was 0.1, the proliferation solution was clear after 8 hours of culture, and the titer was the highest, which indicated that the optimum multiplicity of infection of RDP-CP-22005 was 0.1, and the corresponding highest titer was 7.93X10 9 PFU/mL.
Example 6 determination of one-step growth curve of phage RDP-CP-22005
1. The experimental method comprises the following steps:
Inoculating clostridium perfringens CP-20032 and phage RDP-CP-22005 according to the ratio of optimal infection complex, water-bathing for 10min at 37deg.C, centrifuging for 5min at 12000r/min at normal temperature, discarding supernatant, removing free phage not adsorbed on host, and washing twice with FTG broth. The pellet was resuspended in FTG broth at 37 ℃ and rapidly placed in a shaking table at 37 ℃ for 200r/min for shaking culture and timing. Sampling and counting every 5min for the first 30min, counting every 20min for 30-60min, counting every 30min for 60-300min, taking time as an abscissa, taking a phage titer logarithmic value as an ordinate, drawing a growth curve, obtaining a incubation period and a lysis period of phage, and calculating average lysis amount. Average lysis = phage titer at end of burst/host concentration at initial stage of infection. The results are shown in FIG. 3.
2. Experimental results and analysis:
As can be seen from FIG. 3, there is no significant change in the titer within 50 minutes after infection of the host bacteria by the phage, indicating a latency period of about 50 minutes, a significant increase in the titer of the phage within 50-100 minutes after infection, and a subsequent stabilization, indicating a phage lysis period of about 50 minutes. Through calculation, the phage lysis amount is about 178 PFU/infected cells, which shows that phage has strong lysis and replication capacity.
Example 7 determination of the pH stability of phage RDP-CP-22005
1. The experimental method comprises the following steps:
An exponential phase host bacterial solution of 10 5 CFU/mL was prepared, and the concentration of the phage solution of RDP-CP-22005 was adjusted according to the optimal multiplicity of infection. The pH values of the FTG liquid culture mediums are respectively adjusted to be 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13, phage liquid is taken and added into equal volumes of the FTG liquid culture mediums with different pH values to be uniformly mixed, a culture box is placed at 37 ℃ for 2 hours, 100 mu L of mixed liquid is taken and mixed with equal volumes of bacterial liquid, the titer is measured through a double-layer plate method, and three pH values are parallel. And drawing a phage pH stability curve by taking the pH value as an abscissa and the logarithmic value of phage titer as an ordinate, and determining the pH range of the highest point. The results of phage RDP-CP-22005 are shown in FIG. 4.
2. Experimental results and analysis:
As can be seen from FIG. 4, phage RDP-CP-22005 maintained good activity in the pH range of 2-13, phage maintained high potency, and phage potency was reduced by only one order of magnitude at pH2 or 13. As the pH value is lowered or raised, the titer of phage is not lowered significantly, indicating that phage RDP-CP-22005 is resistant to strong acids and strong bases.
Example 8 acid and alkali resistance comparison experiment of phage RDP-CP-22005
1. The experimental method comprises the following steps:
Meanwhile, phages RDP-CP-22005 and RDPCP23017 are prepared, wherein the phage RDPCP23017 is preserved with the preservation number of CGMCC N0.45574.
An exponential phase host bacterial solution of 10 5 CFU/mL was prepared, and the phage solutions of RDP-CP-22005 and RDPCP23017 were adjusted in concentration according to the optimal multiplicity of infection. And (3) regulating the pH values of the FTG liquid culture mediums to be 3,5, 7, 9, 11 and 13 respectively, taking phage liquids of RDP-CP-22005 and RDPCP23017, respectively adding the phage liquids into equal volumes of the FTG liquid culture mediums with different pH values, uniformly mixing, standing in a 37 ℃ incubator for 2 hours, and respectively measuring the titers of the two groups of phages. The results of acid and base resistance comparisons of two different phages, phages RDP-CP-22005 and RDPCP23017, are shown in FIG. 5.
2. Experimental results and analysis:
as can be seen from the results of FIG. 5, the phage RDP-CP-22005 can maintain better titer under the environment of pH3-13, while the phage titer of the comparative phage RDPCP23017 is obviously reduced under the environment of strong acid and strong alkali, so that the tolerance of phage RDP-CP-22005 to acid and alkali is obviously stronger than that of phage RDPCP23017.
Example 9 determination of optimal growth temperature for phage RDP-CP-22005
1. The experimental method comprises the following steps:
Preparing 10 5 CFU/mL exponential phase host bacterial liquid, diluting RDP-CP-22005 phage stock solution to a proper concentration according to optimal infection complex number, mixing 100 mu L with an equal volume bacterial liquid, pouring a double-layer flat plate, respectively placing the double-layer flat plate at 30 ℃,40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃ for culturing for 30min and 60min, taking out phage liquid every 10min after 30min, cooling to room temperature, and measuring phage titer by a double-layer plate method, wherein three temperatures are parallel. And drawing a phage-to-temperature sensitivity curve by taking the temperature as an abscissa and taking the logarithmic value of phage titer as an ordinate, and determining the temperature range of phage RDP-CP-22005. The results are shown in FIG. 6.
2. Experimental results and analysis:
as can be seen from FIG. 6, the phage RDP-CP-22005 can maintain 80% of activity below 50 ℃, can maintain the activity above 60min at 70 ℃, and can rapidly decrease in 60min in the 90 ℃ environment, which indicates that the phage can withstand a certain high temperature and cannot withstand extremely high temperature.
Example 10 determination of the genetic stability of phage RDP-CP-22005
1. The experimental method comprises the following steps:
Phage RDP-CP-22005 was serially passaged 30 times, equal amounts of RDP-CP-22005 and CP-20032 were added to each culture medium, each culture time was 6 hours, and then the plates were inverted and titers were determined. The results are shown in FIG. 7.
2. Experimental results and analysis:
As can be seen from FIG. 7, the phage RDP-CP-22005 always maintains higher titer in the process of passage 30 times, and no obvious fluctuation appears, which indicates that the phage can be stably inherited and is suitable for industrial production.
EXAMPLE 11 phage lysis-spectrum assay
1. The experimental method comprises the following steps:
Phage host profile was detected by spot method. Randomly selecting host 44 strains of clostridium perfringens with different serotypes from a bacterial library, respectively coating 100 mu L of bacterial liquid to be detected on a culture medium of anaerobic jerky Shang Guti, after airing, absorbing 2 drops of 10 mu L of phage liquid on the culture medium, carrying out anaerobic inversion culture at 37 ℃ for 12-16 hours, observing whether plaque appears, and if so, indicating that the phage has a lysis effect on the strain.
2. Experimental results and analysis:
As shown in Table 4, phage RDP-CP-22005 had a lytic effect on 40 strains of 44 Clostridium perfringens selected in the test, which were derived from the applicant's laboratory and isolated by themselves, both of chicken origin. The lysis rate was 90.9%, indicating a broad lysis spectrum of the phage. The results are shown in Table 4.
TABLE 4 phage RDP-CP-22005 cleavage Spectrum
As can be seen from Table 4, the phage has a better lysis effect on clostridium perfringens, can lyse hosts of various serotypes, has a wider lysis spectrum, can replace antibiotics, and is environment-friendly.
Example 12 chick treatment experiment with phage RDP-CP-22005
1. The experimental method comprises the following steps:
(1) 75 white feather broilers at 1 week old are taken, a test group, a control group and a blank group are arranged, the chickens are randomly divided into 5 groups (15 chickens in each group), each group of chickens in the test group and the control group only takes 1mL clostridium perfringens CP-20032 (10 8 CFU/mL) orally, and the blank group takes an equal volume of physiological saline orally.
(2) The test group is that taking phage RDP-CP-22005, equally dividing into 3 groups, wherein group A is phage RDP-CP-22005 (10 9 PFU/mL) and culture solution with pH=3 are mixed for 2 hours in equal volume, group B is phage RDP-CP-22005 (10 7 pFU/mL) and normal saline, and group C is phage stock solution (10 5 PFU/mL) control group.
(3) After 2h, each of the above test groups was orally administered 1mL of phage from a different group, and the control group and the blank group were intramuscular injected with an equal volume of physiological saline. Grouping and dosing are shown in Table 5.
TABLE 5 phage therapy grouping
(4) The mental status of each group of chickens is observed every day, 8 days are continuously observed, the death condition of the chickens is counted, and a survival curve is drawn. The weights of the chickens in each group before and after the challenge for 8 days are weighed, and the differences of the weights of the chickens in each group are compared. The results are shown in FIG. 8.
2. Experimental results and analysis:
As can be seen from fig. 8, after the experimental chickens are infected with the host, the control chickens have serious death, and within 8 days, the death rate of the chickens is 60% and the death rate of the chickens is 9; the number of deaths of chickens within 5d of phage A, B and C was 0; the number of deaths of chickens in phage A group in 8d was 2, the mortality rate was 13.3%, the number of deaths of chickens in phage B group in 8d was 1, the mortality rate was 6.7%, and the number of deaths of chickens in phage C group and blank group in 8d was 0. That is, phage group A after 2h of treatment in acid environment still has better protection effect on test chickens, phage group B and phage group C also have better protection effect on chickens, tests also prove that phage RDP-CP-22005 has better protection effect on death caused by clostridium perfringens, the phage still has better protection effect after 2h of treatment in acid environment, so that phage has better resistance effect on gastric acid, and cannot be inactivated due to oral administration to alimentary canal, and the phage can effectively prevent clostridium perfringens.
EXAMPLE 13 phage therapy Large group assay
In some local farm in Shandong, there is 5000 chickens in a laying hen house, the feed intake of the chicken flock is reduced, feathers are fluffy, and water sample and thin manure are accompanied by mucus. The clostridium perfringens is detected by a laboratory and is clostridium perfringens enteritis disease.
Phage RDP-CP-22005 broth (effective concentration 10 8 PFU/mL) was waterline fed to the chicken flock. After the phage is used for 24 hours continuously for 3 days, the diarrhea symptom is obviously relieved, the diarrhea phenomenon is basically eliminated after the phage is used for 48 hours, the feed intake is increased, the spirit is good, and the chickens are healed after the phage is used for 72 hours.
The large group of experiments prove that the phage of the invention can treat clostridium perfringens in practical application, has obvious effect, reduces the resistance to the antibody, and is green and has no residue.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (6)
1. The acid and alkali resistant clostridium perfringens bacteriophage RDP-CP-22005 is characterized in that clostridium perfringens bacteriophage is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 45449 in the year 2023 and the month 23.
2. Clostridium perfringens bacteriophage RDP-CP-22005 according to claim 1 wherein said clostridium perfringens bacteriophage has good acid and alkali resistance at a pH of 2.0-13.0.
3. A bactericidal composition comprising clostridium perfringens bacteriophage RDP-CP-22005 according to claim 1.
4. Use of clostridium perfringens bacteriophage RDP-CP-22005 according to claim 1 or of the bactericidal composition according to claim 3 for the preparation of at least one of the following products: (1) a product that kills or inhibits clostridium perfringens; (2) A product for preventing and/or treating diseases of poultry caused by clostridium perfringens; (3) A product for preventing and/or treating inflammatory reactions caused by clostridium perfringens.
5. A phage pharmaceutical formulation, wherein the active ingredient of the phage pharmaceutical formulation comprises clostridium perfringens phage RDP-CP-22005 according to claim 1 or the bactericidal composition according to claim 3.
6. The phage pharmaceutical formulation of claim 5, further comprising a pharmaceutically acceptable carrier; the pharmaceutical preparation is in the form of oral administration or injection administration.
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| CN116042593A (en) * | 2023-01-16 | 2023-05-02 | 山东省农业科学院畜牧兽医研究所 | Clostridium perfringens bacteriophage lyase and application thereof in preparation of clostridium perfringens infection resistant medicines |
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