CN118147090A

CN118147090A - Phage for simultaneously lysing multiple strains of escherichia coli and salmonella and application thereof

Info

Publication number: CN118147090A
Application number: CN202410571160.5A
Authority: CN
Inventors: 赵成新; 刘晓; 刘玉华; 邹德志; 房超; 王官晓; 徐文彬; 王先德; 张炜; 宋玉财
Original assignee: Yantai Jinhai Pharmaceutical Co ltd
Current assignee: Yantai Jinhai Pharmaceutical Co ltd
Priority date: 2024-05-10
Filing date: 2024-05-10
Publication date: 2024-06-07

Abstract

The invention relates to a bacteriophage for simultaneously lysing multiple strains of escherichia coli and salmonella and application thereof, belonging to the technical field of biology; the phage is salmonella phage PSA585, the preservation number is CCTCC NO: M2024026, and the whole genome sequence is shown as SEQ NO. 1-SEQ NO. 8; the salmonella phage PSA585 obtained by the invention can be used for preparing phage products, can be used singly or in a mixed mode, is used for treating bacterial infection caused by salmonella and escherichia coli clinically, or is used as an environmental biological disinfectant for preventing, controlling and purifying salmonellosis and escherichia coli, can lyse escherichia coli and salmonella, has wide lysis spectrum and strong lysis property, solves the problem of narrow host spectrum of single phage, and improves the clinical application effectiveness of phage products.

Description

Phage for simultaneously lysing multiple strains of escherichia coli and salmonella and application thereof

Technical Field

The invention relates to a phage for simultaneously lysing multiple strains of escherichia coli and salmonella and application thereof, belonging to the technical field of biology.

Background

Coli, also known as escherichia coli, is a gram-negative bacterium of the enterobacteriaceae family and is widely found in nature. Pathogenic escherichia coli is one of common pathogenic bacteria in the field of poultry cultivation, can cause local or systemic infection in various poultry, such as chickens, turkeys, ducks and the like, has the highest morbidity in chickens, and especially has the death rate of more than 53.5 percent. Salmonella belongs to enterobacteriaceae, gram-negative bacteria and spore-free intracellular pathogens, can cause various diseases, is a main zoonotic pathogen, and is commonly known as salmonella enteritidis, salmonella typhimurium and salmonella pullorum. Salmonella can be divided into 2600 more serotypes based on lipopolysaccharide O-antigen and flagella H-antigen, with salmonella enteritidis of the O9 serotype being more prevalent. Along with the continuous expansion of chicken raising scale, the infection incidence rate of chicken-origin salmonella is also in an ascending trend, mixed sense can sometimes appear, and common escherichia coli mixed sense salmonella is mostly seen, and for a long time, the treatment of chicken colibacillosis and salmonellosis mainly depends on antibiotics, so that bacterial drug resistance is aggravated, the effect of clinically using antibiotics for treating the escherichia coli disease and the salmonellosis is poorer and worse, and the clinical mixed sense further causes the difficulty of drug treatment to be increased. Phages are a class of viruses that are parasitic in bacteria, fungi, actinomycetes and spiral phages, affecting the evolution of most microbial species, and are one of the important factors controlling bacterial death. The phage can specifically and efficiently lyse host bacteria, and the phage is used for treating diseases, and compared with antibiotic therapy, the phage has the advantages of low minimum bacterial killing dosage, high specificity, high sterilization effect, high screening speed, quick response, safety, no side effect and the like. Phage is a natural antibacterial agent that induces bacterial lysis, maintains flora balance, and has a mechanism of action that is completely different from antibiotics, and has effects on both G+, G-and drug-resistant bacteria. Phage are widely distributed, can penetrate through the blood brain barrier, can be used for treating central nervous system infection, and has far lower treatment cost than antibiotics.

Phage were originally used to treat intestinal infections in livestock and poultry. In recent years, research shows that the phage preparation can be added into feed to obviously improve the growth performance of poultry and improve the intestinal flora structure of broilers. Easwa-ran and the like successfully isolate escherichia coli lytic phage by taking escherichia coli as host bacteria, and compare the escherichia coli treated by phage with an untreated escherichia coli growth curve through experiments, so that the phage can effectively inhibit the escherichia coli from growing and treat colibacillosis. Kim and the like are added into the feed of broiler chickens, and the probability of the broiler chickens eating the salmonella enteritidis phages for promoting the growth of the broiler chickens suffering from salmonella enteritidis is greatly reduced. T.H.Lim, etc. have good effects on treating infection of layers caused by salmonella by using phage, and the mortality rate of the layers is only 5% when the phage is used as a feed additive for treating infection of layers, and the mortality rate of the layers which are not treated by phage is 30%. Zhuo Guorong and the like select 672 1-day-old broilers of about 45g, 0.25g/kg and 0.5g/kg phage are respectively added into the feed, the growth performance, blood characteristics and microbial shedding of the broilers are observed, and the result shows that 0.50g/kg phage can improve the early feed conversion rate and the relative weight of liver of the broilers, increase the quantity of intestinal lactobacillus and reduce the quantity of intestinal escherichia coli and salmonella. Kittler and the like select 64 broilers, and phage preparations are added into drinking water, and the result shows that phage can effectively reduce the quantity of intestinal escherichia coli.

However, most phages have strict host specificity and a narrow host spectrum, and a phage of one type or species can only infect bacteria of the corresponding type or species. Different phage host spectra and biological characteristics are different, and generally, the single phage has lower lytic property and coverage rate. The phage which can crack salmonella and colibacillus is screened to be used for clinical phage preparation research, the phage cracking spectrum can be widened, and the problem of mixed sense of clinical colibacillus and salmonella is solved.

Disclosure of Invention

The invention aims to provide a phage capable of simultaneously lysing a plurality of escherichia coli and salmonella, and the phage is applied to laying hen breeding, so that the escherichia coli and the salmonella are prevented and controlled, and particularly the loss of the escherichia coli and the salmonella to the breeding industry is effectively reduced aiming at the clinical mixed sense problem.

The technical scheme of the invention is as follows:

the invention provides a phage which lyses multiple strains of escherichia coli and salmonella simultaneously, the phage is named salmonella phage PSA585, and the phage has been submitted for bacterial preservation.

[ Biological preservation Specification ]

Preservation unit: china center for type culture Collection;

preservation address: chinese armed chinese;

preservation date: 2024, 01, 05;

Preservation number: CCTCC NO: M2024026;

Classification naming: salmonella sp.phagePSAS5.

Further, the whole genome sequence of the phage is shown as SEQ NO. 1-SEQ NO. 8.

The invention also discloses application of the phage capable of simultaneously lysing multiple strains of escherichia coli and salmonella in preparing a medicament for treating and/or preventing diseases caused by salmonella and/or escherichia coli.

The invention also discloses a bactericidal composition for preventing and treating salmonella and escherichia coli, which comprises phage capable of simultaneously lysing a plurality of escherichia coli and salmonella.

The invention also discloses an environment biological disinfectant, which comprises phage capable of simultaneously lysing multiple strains of escherichia coli and salmonella.

Further, the environmental biological disinfectant is used for controlling and/or purifying salmonella and/or colibacillosis.

The invention also discloses application of the phage capable of simultaneously lysing a plurality of escherichia coli and salmonella in laying hen culture. After the phage attacks escherichia coli and salmonella, the protection rate of the phage to chickens reaches 85% or more, and the phage can be applied to laying hen breeding, so that the loss of escherichia coli and salmonella to the breeding industry is effectively reduced.

Compared with the prior art, the invention has the following beneficial effects:

Salmonella phage PSA585 of the present invention can cleave both E.coli and Salmonella. The cracking rate of the separated salmonella host strain reaches 77.5%, and when the salmonella host strain is used alone, the salmonella host strain has relatively strong cracking capacity and a relatively wide cracking spectrum; the microbial preparation also has broad-spectrum cracking capability to escherichia coli, and the cracking rate reaches 71.91%. Solves the problem of narrow host spectrum of single phage, and improves the clinical application effectiveness of phage products.

The salmonella bacteriophage PSA585 can be used for preparing a bacteriophage preparation by taking salmonella JHSA as a host bacterium for fermentation and is used for clinic; optimal culture conditions were determined by exploring optimal liquid proliferation conditions such as temperature, pH, MOI, etc. of salmonella phage PSA585, and optimal preparation conditions of phage were determined by stepwise amplification culture.

The salmonella bacteriophage PSA585 of the present invention can be used to prepare phage products, alone or in combination, for treating bacterial infections caused by salmonella and escherichia coli clinically, or as an environmental biological disinfectant for controlling and purifying salmonellosis and escherichia coli.

Drawings

FIG. 1 is an electron microscope image of a Salmonella phage PSA585 of the present invention;

FIG. 2 is a genomic map of Salmonella phage PSA585 of the present invention;

FIG. 3 is a one-step growth plot of Salmonella phage PSA585 of the present invention;

FIG. 4 is a graph of the viability of Salmonella phage PSA585 of the present invention at different temperatures;

FIG. 5 is a graph of the survival rate of Salmonella phage PSA585 of the present invention at different pH's.

Detailed Description

The principles and features of the present invention are described below in connection with examples, which are set forth only to illustrate the present invention and not to limit the scope of the invention.

The media, reagents and equipment and materials used in the following examples were:

LB (Luria broth) liquid Medium (1L): 10g of peptone, 5g of yeast powder, 110g of NaC, adding ddH20 to 1L, adjusting the pH to 7.0,121 ℃ and sterilizing for 20 min.

0.6% LB semisolid medium (1L): 10g of protein, 5g of yeast powder, 1 g of NaC, 6g of agar powder, adding ddH20 to 1L, adjusting the pH to 7.0,121 ℃ and sterilizing under high pressure for 20 min.

1.2% LB solid Medium (1L): 10g of peptone, 5g of yeast powder, 10g of NaCl, 12g of agar powder, adding ddH20 to 1L, adjusting the pH to 7.0,121 ℃, performing high-pressure sterilization for 20min, cooling to 50 ℃, pouring the mixture into a flat plate, solidifying and inverting for later use.

SS medium (1L): 5.0g of peptone, 5.0g of beef extract powder, 8.5g of ox gall salt, 10.0g of lactose, 8.5g of sodium citrate, 8.5g of sodium thiosulfate, 1.0g of ferric ammonium citrate, 0.025g of neutral red, 0.00033g of brilliant green, 16.0g of agar, 20 to 1L of ddH, stirring, boiling and dissolving for 1 minute, adjusting the pH value to 7.0, cooling to 50 ℃, pouring the flat plate, and inverting for later use after solidification.

Wheat Conkai medium (1 liter): weighing 50.0g of the Maiconkai culture medium in 1L of distilled water or deionized water, boiling to dissolve completely, packaging, sterilizing at 121deg.C under high pressure for 15 min, and shaking to prevent agar from depositing at the bottom of the vessel and solidifying.

SM buffer: accurately weighing 5.8g of NaC1,2g of MgS0 ₄·7H₂, dissolving in 800mL of double distilled water, adding 50mL of 1mol/L Tris-HC1 (pH 7.5), adding distilled water to 1000mL, sterilizing at 121 ℃ for 15min, and preserving at room temperature.

50% LB glycerol frozen stock: after 100ml of LB liquid medium and 100ml of glycerol are mixed uniformly, the mixture is autoclaved at 121 ℃ for 20min and then refrigerated at 4 ℃.

2% Phosphotungstic acid (PTA), PEG8000, naCl, DNaseI, RNaseA, chloroform and other reagents are commercially available.

Equipment and materials: a vertical pressure steam sterilizer LDZF-75L (Shanghai Shenan technologies Co., ltd.); biological safety cabinet (Beijing Dongbi Harr instruments Co., ltd.), LRH-500F constant temperature incubator (Shanghai-Heng scientific instruments Co., ltd.), constant temperature shaking table HYL-C2 (Taiku Kung Hai Leu laboratory equipment Co., ltd.), TGL-16M low temperature high speed refrigerated centrifuge (Changsha Hunan instruments centrifuge instruments Co., ltd.), micropipette, culture dish, disposable syringe and 0.22 μm, 0.45 μm microporous needle filter, culture dish, triangular flask, etc.

EXAMPLE 1 separation of Salmonella

Collecting disease materials from poultry farms of Anhui, hainan Haikou, liaoning Benxi, guangdong Kaiping, hebei Xingtai and Shandong plummet Mouping, taking diseased bird heads and livers by aseptic operation, streaking on a selective culture medium (SS agar), culturing at 37 ℃ for 18-24 hours, wherein salmonella colonies are brown or black, have a diameter of about 2-3 mm, have a rough surface and irregular edges. The typical single colony was picked up and streaked for purification 3 times, and then the single colony was picked up and inoculated into 5mL of LB broth, and cultured for 8 hours at 37℃under 180rpm with shaking, to give a uniform turbid bacterial suspension. And then through 16s RNA molecular identification and serotype identification, the separated strains are determined to be pathogenic salmonella, and are respectively named JHSA585, JHSA080, JHSA081, JHSA082, JHSA083, JHSA084, JHSA085, JHSA086, JHSA087, JHSA088 and JHSA089, and the strains are stored in a refrigerator at the temperature of minus 80 ℃ by using LB glycerol frozen stock.

EXAMPLE 2 isolation of E.coli

Sampling the disease materials from Shandong tobacco stand Mouping, tobacco stand Laiystate, shanxi Taiyuan, liaoning Benxi, shandong Laiyang, shandong Zhucheng, hebei Xingtai, shandong Qingzhou, liaoning Yingkou, jilin Changchun and Guangdong Kai flat poultry farm, taking the disease heads and livers through aseptic operation, taking the disease materials through a sterilized inoculating loop, streaking and inoculating on a Maiconkai agar medium, pouring the medium into a 37 ℃ incubator for culturing for 18-24 hours, then taking red colonies conforming to the culturing characteristics of escherichia coli, and detecting the gram-negative medium-sized bacillus through a smear color dyeing mirror. Single colony is picked up and inoculated in LB solid medium for pure culture, and then is subjected to streak purification for 3 times, and then single colony is picked up and inoculated in 5mL LB broth, and is subjected to shaking culture for 8 hours at 37 ℃ at 180rpm, so that uniform and turbid bacterial suspension is obtained. And then through 16s RNA molecular identification and serotype identification, the separated strains are determined to be pathogenic escherichia coli, and are respectively named JHEC001、JHEC002、JHEC003、JHEC004、JHEC005、JHEC006、JHEC007、JHEC008、JHEC009、JHEC011、JHEC012、JHEC013、JHEC014、JHEC015、JHEC016、JHEC017、JHEC018、JHEC019、JHEC020、JHEC021、JHEC022、JHEC023、JHEC024、JHEC025、JHEC026、JHEC027, and stored in a refrigerator at-80 ℃ by using LB glycerol frozen stock.

EXAMPLE 3 isolation, purification and preservation of Salmonella phage PSA585

Phage isolation: the sample is taken from sewage in a chicken farm septic tank of Shandong Zhucheng, the sample is centrifuged for 10min at 4 ℃ and 12000 rpm, the supernatant is centrifuged again for 3 times, and the final supernatant is filtered by a filter membrane of 0.45 μm and a filter membrane of 0.22 μm respectively; taking 5mL of filtrate, adding JHSA 585.1 mL of salmonella which is separated and stored, adding 5mL of 2xLB liquid medium, placing at 37 ℃ for culturing for 14-16 h, centrifuging the culture at 4 ℃ for 10min at 12000 rpm the next day, filtering and sterilizing the supernatant by using a 0.22um filter membrane, thus obtaining stock solution containing phage, namely phage suspension.

Phage purification: 100. Mu.L of the phage suspension and 100. Mu.L of salmonella JHSA in logarithmic growth phase bacterial suspension are added into a 20mL sterilization centrifuge tube by a pipette, allowed to stand for infection for 15min, then 8mL of LB semisolid culture medium containing 6g/L agar cooled to 50 ℃ is added, uniformly mixed, poured onto a pre-coagulated LB plate to prepare a double-layer plate, and the double-layer plate is cultured at 28 ℃ in an inverted manner overnight. Selecting single plaques, diluting with 1ml SM buffer solution in a multiple ratio, filtering and sterilizing phage diluent with a 0.22 mu m filter membrane, mixing 100 microliters of phage diluent with 100 microliters of salmonella JHSA585 bacterial suspension in logarithmic growth phase, adding 8ml of LB semisolid culture medium, mixing, pouring onto a pre-coagulated LB plate to prepare a double-layer plate, inversely culturing overnight at 28 ℃, repeating the above operation for 3-5 times, and purifying phage until the sizes and shapes of plaques are basically consistent, the diameters of plaques on the double-layer plate are 2-4 mm, and the periphery of the plaques are clear and have no halo, so that the phage is a typical lytic phage.

Phage morphology: the purified phage suspension is used for electron microscope observation, 20uL phage suspension containing crude phage particles is dripped on a copper mesh, natural precipitation is carried out for 15min, redundant liquid is sucked off from the side face by filter paper, a drop of 2% phosphotungstic acid (PTA) is added on the copper mesh, the phage is dyed for 10min, then the filter paper is used for sucking off the dyed liquid from the side face, after the sample is dried, the shape of the phage is observed by an electron microscope, as shown in figure 1, the head of the obtained phage is in a regular icosahedron, the nucleic acid is wrapped, the diameter of the phage is about 56nm, the tail is about 110nm, and the neck is connected with the head and the tail. According to the ninth report of International taxonomic tissue Virus Classification, the phage can be classified as having a Rheumatoid order of Rheumatoid family, designated Salmonella phage PSA585.

Phage enrichment cryopreservation: the single plaque in the double plate is picked up in 1ml SM buffer solution, 100 mu L phage solution is mixed with 100 mu L salmonella JHSA separated in example 1, 8ml LB semisolid culture medium is added, after mixing, the mixture is poured onto the pre-coagulated LB culture medium to prepare a double plate, the double plate method is used for culturing, 5ml SM buffer solution is added into the plate until the plate forms a transparent empty plate, and the plate is shaken for 2-4 hours at 4 ℃ and 60 rpm. Taking SM solution in a flat plate, centrifuging 4000g for 5min, filtering by a 0.22 mu m inlet filter, freezing and storing the filtered phage enrichment solution and LB glycerol frozen stock solution according to the ratio of 1:1, and storing in a refrigerator at the temperature of minus 80 ℃.

Phage PEG purification and cold preservation: adding DNaseI, RNaseA to the phage lysate to a final concentration of 1 μg/ml, incubating for 30min at 37 ℃, adding NaCl ice bath with a final concentration of 1M for 1h, centrifuging for 10min at 4 ℃ and 11000g, taking the supernatant, adding PEG8000 with a final concentration of 10%, overnight at 4 ℃, centrifuging for 10min at 4 ℃ and 11000g, discarding the supernatant, inverting for 5min, removing excessive water as far as possible, adding SM buffer to the rest solid matters for resuspension, adding an equal volume of chloroform and shaking 30s gently, centrifuging for 15min at 4 ℃ and 3500g to separate an organic phase and a hydrophilic phase, recovering the hydrophilic phase containing phage particles, split charging the purified phage suspension into a 2ml centrifuge tube, and storing in a refrigerator at 4 ℃.

The salmonella phage PSA585 obtained by separation and purification is subjected to whole genome sequencing, and the sequence is shown as SEQ NO. 1-SEQ NO. 8. The genome map of this salmonella phage PSA585 is shown in fig. 2, the whole genome sequence size of phage PSA585 is 85374 bp, the gc content is 49.5%, and the total contains 118 ORFs (open reading frames) which encode phage DNA precursor components and putative proteins, respectively.

Example 4 Salmonella phage PSA585 potency assay

(1) Preparing a bacterial suspension of host bacteria JHSA: taking out the preserved strain JHSA from the refrigerator at-80 ℃, streaking and inoculating on a Maiconka agar plate, culturing for 24 hours at 28 ℃, then picking a single colony and inoculating in an LB liquid culture medium, and adjusting the concentration of bacterial liquid to 1X 10 ⁹ CFU/ml in the same way.

(2) Phage preservation solution gradient dilution: taking sterilized 1.5 ml centrifuge tubes, respectively adding 900 microliter SM buffer solution into a first tube, adding 10 microliter phage stock solution into a second centrifuge tube after uniformly mixing, adding 10 microliter phage stock solution into a third centrifuge tube after uniformly mixing, and so on, carrying out gradient dilution, taking 100 microliter phage diluent of each gradient and 100 microliter host bacteria solution, fully mixing uniformly, paving double-layer agar plates, culturing at the constant temperature of 28 ℃ for about 10 hours, carrying out plaque counting on each agar plate, selecting plates with 30-300 plaques, calculating the initial concentration of the obtained phage according to dilution factor to obtain phage titer, wherein the phage titer (PFU/ml) =dilution factor×plaque number×10, and the phage titer is 1×10 ⁸ PFU/ml.

Example 5 determination of the optimal multiplicity of infection (MOI) of Salmonella phage PSA585

100. Mu.L of phage multiplication solution and 100. Mu.L of salmonella JHSA bacterial solution separated in example 1 are added into a centrifuge tube containing 800. Mu.L of LB liquid medium according to concentration ratios of 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 and 0.000001 respectively, the mixture is cultured for 5 hours at 28 ℃ and 180rpm, after the culture is finished, the mixture is centrifuged for 5 minutes at 11000g, and the supernatant is collected and filtered to obtain phage filtrate. The phage titers at the different infectious complex numbers were determined by dilution of the filtrate at a multiple ratio (determination of titers in the same manner as in example 4). The initial bacterial liquid concentration is 1X 10 ⁸ CFU/ml, and the phage titer is 1X 10 ⁸ PFU/ml. The results are shown in Table 1.

TABLE 1 determination of optimal multiplicity of infection (MOI) for Salmonella phage PSA585

Phage titer = number of phage plaques x dilution x 10

From the results in Table 1, according to the optimal infection complex number of 1:100, the phage cost performance is ideal, the phage liquid is amplified and cultured for 5 hours, the phage is clear and transparent, the phage titer is high, and the results can provide reference basis for actual production fermentation.

Example 6 one-step growth Curve determination of Salmonella phage PSA585

The phage one-step growth curve refers to the curve drawn by recording phage growth rules, and the incubation period, the lysis period and the rest period of phage can be clearly seen. Adding the above-mentioned standby host bacteria culture JHSA (1×10 ⁸ CFU/ml) and the above-mentioned phage culture solution PSA585 (1×10 ⁸ PFU/ml) into LB liquid culture medium according to optimum MOI (1:100), centrifuging at 28deg.C in thermostatic water bath for 15min,11000g/min for 5min, taking precipitate, washing 2 times with 28 deg.C LB liquid culture medium, taking centrifuged precipitate, adding 28 deg.C LB liquid culture medium for 10ml, mixing, culturing at 28deg.C in thermostatic shaking table for 180r/min, collecting liquid titer every 10min from 0min until 120min, repeating for 12 times each independent experiment for 3 times, and taking average value. The log of phage titer is taken as an abscissa and the log of phage titer is taken as an ordinate, a one-step growth curve is drawn, and the result is shown in figure 2, wherein the titer does not change obviously within 10min after the phage infects host bacteria, which indicates that the incubation period is about 10min, the phage titer rises obviously within 10-110 min after the phage infects, and then the phage titer tends to be stable, at the moment, the phage titer is 3.9 x 10 ⁹ PFU/mL, which indicates that the phage lysis period is about 110min, and the lysis amount is 39PFU/cell. Salmonella phage PSA585 has the characteristics of short latency, strong lytic power and high replication.

EXAMPLE 7 Salmonella phage PSA585 biological Properties

(1) Stability of Salmonella phage PSA585 at different temperatures

Under aseptic conditions, 6 1.5ml aseptic centrifuge tubes were taken and 900. Mu.L of LB liquid medium was added, respectively. 6 sterile centrifuge tubes were preheated in water baths at 30, 40, 50, 60, 70 and 80℃for 60min, after the preheating was completed, 100. Mu.L of phage with a titer of 1X 10 ⁸ PFU/ml were added under sterile conditions, the water bath was continued, and after 60min, the samples were diluted in a doubling ratio and the phage titer was determined by plating a bilayer plate with the salmonella JHSA strain solution isolated in example 1.

The results are shown in FIG. 3: the salmonella phage PSA585 survived at 70℃for 60min at different temperatures. The survival rate can reach more than 60 percent within the range of 30-40 ℃, and when the temperature is increased to 50 ℃, the survival rate can only be kept within the range of 0-10 percent. The optimum survival temperature of the phage is 30-40 ℃.

(2) Stability of salmonella phage PSA585 at different pH

The pH values of 12 liquid media containing 900. Mu.LLB were adjusted to 1,2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, respectively, and 100. Mu.L of Salmonella phage PSA585 at a concentration of 1X 10 ⁷ PFU/ml was added, respectively. Phage titers were measured after 60min pre-heating in a 28 ℃ thermostat water bath, as shown in figure 4, salmonella phage PSA585 was cultured in different pH media for 60min at pH 7 with the highest survival rate and almost complete survival, so the phage had an optimal pH of 7. Phage were inactivated at pH values of 0-2 or 12. The survival rate of the salmonella bacteriophage PSA585 is between 40% and 60% at the pH value of 3-4, the survival rate is more than 60% at the pH value of 5-9, and the survival rate is reduced to more than 20% -50% at the pH value of 10-11.

Example 8 Salmonella phage PSA585 cleavage Spectrum assay

89 Strains of salmonella and 89 strains of pathogenic escherichia coli separated from regions such as Hainan, jiangsu, anhui, shandong, hebei and the like are selected as host bacteria, and a phage lysis spectrum is measured by a spot method, and a lysis rate is counted.

(1) Resuscitates the host bacteria, inoculates 100 mu L of host bacteria in 100ml of LB liquid medium, and cultures the host bacteria for 8 hours at 37 ℃ under 180rpm shaking.

(2) 100 Mu L of the host bacteria liquid is coated on an LB solid plate, and the names of the host bacteria are marked on the plate, so that confusion is avoided.

(3) 3.5 Mu L of salmonella phage PSA585 is dripped in the center of a flat plate, fully dried, inverted and subjected to stationary culture in a constant temperature incubator at 30 ℃ for 8-10 hours. Plaques were observed, and the clear transparency of plaques was used as an evaluation criterion for the intensity of phage lysis.

(4) The extent of lysis was assessed in terms of plaque clarity, "-" indicates no lysis; "+" indicates cleaved but opaque; "++" indicates cleaved and clear.

The results are shown in Table 2:

TABLE 2 determination of the Salmonella phage PSA585 cleavage Spectrum

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From the aspect of phage lysis, the (-) of the sterile plaques and (+) -of the opaque plaques are discarded, and the result of the (+ +) data of the transparent plaques is selected for statistical analysis, so that the salmonella phage PSA585 has a salmonella lysis rate of 77.5 percent and a salmonella lysis rate of 71.91 percent, which indicates that the salmonella phage PSA585 has a high salmonella lysis rate and a good salmonella lysis effect on escherichia coli, and the phage has a wide lysis spectrum and strong lysis property, and is very suitable for being used alone or for preparing phage cocktails.

Example 9 evaluation of clinical Effect of Salmonella phage PSA585 on Salmonella gallinarum and E.coli

150 Chickens of 12 days old are selected for test and divided into A, B, C groups, 50 chickens in each group are divided into A, B, C groups, A group is an escherichia coli test group (strain JHEC 008), B group is a salmonella test group (strain JHSA 585), C group is escherichia coli+salmonella test group, each group is divided into 5 groups (such as A1, A2, A3, A4 and A5), each group is divided into 10 chickens, 4 groups are subjected to virus attack pathogenic bacteria, 1 group is not subjected to virus attack and is used as negative control, 2 groups are selected after virus attack and treated with salmonella bacteriophage PSA585, and 2 groups are subjected to bacteriophage attack and are used as test groups. As shown in table 3, the mortality and protection rate of chicks were counted.

Table 3 clinical efficacy evaluation of salmonella phage PSA585 in the treatment of salmonella gallinarum and escherichia coli

As can be seen from Table 3, after the salmonella phage PSA585 attacks escherichia coli and salmonella, the chick protection rate can reach 85% or more, and the application effect is good.

Example 10 fermentative preparation of Salmonella phage PSA585

(1) Preparing fermentation seed liquid: and inoculating JHSA585 100 mu L of preserved salmonella into 300ml of LB liquid medium, and carrying out shaking culture at 180rpm on a shaking table at 37 ℃ for 8 hours to obtain host bacterial suspension. Mixing Salmonella phage PSA585 100 μL with 100 μL Salmonella JHSA585 host bacteria, standing at room temperature for 15min to allow phage to infect and adsorb host bacteria cells as much as possible, adding 100ml LB liquid medium, shaking at 28deg.C and 180rpm for 5 hr, centrifuging 4000g of obtained phage culture liquid for 15min, and preserving at 4deg.C for use.

(2) Phage production fermentation: the salmonella phage PSA585 fermentation preparation system is 20L, and the fermentation medium is LB medium. Inoculating by flame inoculation, and respectively inoculating 30ml phage PSA585 (10 ⁸ PFU/ml) and 300ml host bacteria JHSA bacterial liquid (10 ⁸ CFU/ml) into a fermentation culture medium according to the corresponding optimal infection complex ratio (1:100). Sterile air is introduced in the fermentation process, 3% of defoamer is added, and the fermentation time is 4 hours. 20ml of a mixed solution of phage and host bacteria is taken every 1h from the beginning to the end of fermentation, the mixed solution is placed in a 25ml sterile centrifuge tube, 4000g is centrifuged for 15min, supernatant fluid is taken and sterilized by a microporous filter membrane with 0.22um to obtain a filtrate containing phage, and then the titer is measured after dilution by using SM buffer solution in a multiple ratio, and the titer is shown in table 4.

TABLE 4 Salmonella phage PSA585 production fermentation potency assay

As can be seen from Table 4, salmonella phage PSA585 was most aged at 6.9X10 ¹¹ PFU/ml during 3h of fermentation. After 4 hours fermentation, the phage titer is increased from the initial 10 ⁸ PFU/ml to more than 10 ¹¹ PFU/ml, which is improved by 3 orders of magnitude. Large scale industrial preparation of phage can be performed using fermentation methods.

The above embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention, but any modifications, equivalents, improvements, etc. within the spirit and principles of the present invention should be included in the scope of the present invention.

Claims

1. A phage for simultaneously lysing a plurality of escherichia coli and Salmonella is characterized in that the phage is preserved in China Center for Type Culture Collection (CCTCC) in 1 month and 5 days of 2024, the preservation number is M2024026, the preservation address is Chinese Wuhan, and the phage is classified and named as Salmonella sp.phage PSA585.

2. The bacteriophage which lyses simultaneously a plurality of escherichia coli and salmonella according to claim 1, wherein the whole genome sequence of the bacteriophage is shown in SEQ No.1 to SEQ No. 8.

3. Use of a bacteriophage which lyses simultaneously multiple strains of escherichia coli and salmonella as claimed in claim 1 or 2 for the preparation of a medicament for the treatment and/or prophylaxis of diseases caused by salmonella and/or escherichia coli.

4. A bactericidal composition for controlling salmonella and escherichia coli comprising a bacteriophage which lyses a plurality of escherichia coli and salmonella simultaneously according to claim 1 or 2.

5. An environmental biological disinfectant comprising a bacteriophage of claim 1 or 2 that lyses multiple strains of escherichia coli and salmonella simultaneously.

6. An environmental biological disinfectant according to claim 5, for controlling and/or purifying diseases caused by salmonella and/or escherichia coli.

7. Use of a bacteriophage which lyses simultaneously multiple strains of escherichia coli and salmonella as claimed in claim 1 or 2, in laying hen breeding.