CN117070471A - Multivalent salmonella phage capable of entering blood orally and application thereof - Google Patents
Multivalent salmonella phage capable of entering blood orally and application thereof Download PDFInfo
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- 238000000855 fermentation Methods 0.000 abstract description 19
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Abstract
The invention relates to the field of application of microbiological technology, and particularly discloses a multivalent salmonella bacteriophage capable of entering blood orally and application thereof, wherein the preservation number of the bacteriophage is CCTCC NO: m20231500. The bacteriophage SAP56 in the invention has high fermentation efficiency, and the host bacteria OD 600 At a final phage inoculation concentration of 2.3X106 PFU/mL, phage SAP56 titers were highest, up to 5.7X10 11 PFU/mL. The oral administration can enter blood, can effectively prevent and control systemic infection caused by salmonella and escherichia coli, and provides a biological prevention and control measure which is safe, has no toxic or side effect and can be produced by large-scale fermentation for salmonella and escherichia coli.
Description
Technical Field
The invention relates to the field of application of microbiological technology, in particular to a multivalent salmonella bacteriophage capable of entering blood orally and application thereof.
Background
Salmonellosis (Salmonellosis) refers to an acute or chronic disease in an avian caused by any one or more members of the Salmonella genus. Salmonella is a member of the vast Enterobacteriaceae family, and Salmonella comprises 2100 serotypes. Poultry constitutes the largest individual storage host for salmonella in nature. Among all animals, the most commonly reported salmonella is derived from poultry and poultry products, as are human salmonella infections and food poisoning. With the rapid development of the poultry industry, salmonella-induced salmonellosis of poultry is widely spread, making it one of the most important egg-borne bacterial diseases of poultry. However, in the poultry breeding process, the immunity of organisms is reduced due to stress caused by external factors such as climate, human beings and the like, so that various bacterial diseases are secondary, wherein the mixed sensing condition of salmonella and escherichia coli frequently occurs, and the prevention and control effects on the bacterial diseases are also much worse due to the antibiotic resistance.
Phage are a beneficial virus which specifically attacks bacteria, widely exist in nature, are safe to use, have no toxic or side effect, and specifically lyse pathogenic bacteria without damaging intestinal flora. The phage can effectively lyse pathogenic bacteria, has great advantages and potential in preventing and treating bacterial diseases in poultry cultivation, and is a biological prevention or treatment means with application prospect.
Application CN202310170864.7 discloses application of phage composition in slaughter houses, which can prevent salmonella infection and escherichia coli infection and ensure safety of livestock, poultry and the like in slaughter processes.
Application CN112680423A discloses a broad-spectrum strong-lytic coliphage capable of simultaneously lysing four bacteria, and the phage not only has a good lysis effect on colibacillus, but also can lyse shigella, salmonella and enterobacter cloacae.
The application CN202211231920.5 discloses a salmonella phage nct and application thereof, wherein the phage is a broad-spectrum multivalent salmonella phage capable of lysing 3 strains of salmonella enteritidis, 15 strains of escherichia coli and 10 strains of klebsiella pneumoniae, and provides a novel prevention and treatment means for environmental and food pollution and the like caused by salmonella and escherichia coli.
Although there are studies on multivalent salmonella phages and salmonella phages for controlling salmonella infection, there is no report of multivalent salmonella phages entering the blood. If phage cannot enter blood orally, it cannot reach target organs, and thus cannot work well against systemic infections caused by salmonella and/or escherichia coli.
The salmonella phage SAP56 obtained by screening can crack salmonella, has a strong cracking effect on pathogenic escherichia coli separated clinically, can enter blood to directly reach focus, and provides a novel biological prevention and control method for tissue mixed infection caused by salmonella and escherichia coli.
Disclosure of Invention
The invention aims to provide a salmonella bacteriophage, and the preservation number of the bacteriophage is CCTCC M20231500.
Another object of the invention is to provide the use of salmonella phage for controlling mixed infection of salmonella and escherichia coli.
In order to achieve the above object, the present invention adopts the following technical measures:
the applicant screens out a salmonella phage from water samples near a chicken farm in Hubei province, and the phage is sent to China center for type culture collection for preservation on the day 17 of 08 in 2023, and the phage is named after classification: salmonella phage SAP56, address: university of martial arts, deposit number: cctccc NO: m20231500.
The phage has morphological characteristics of conventional salmonella phage, belongs to multivalent phage, and can simultaneously lyse salmonella and escherichia coli.
The protection scope of the invention comprises:
use of Salmonella phage SAP56 for the manufacture of a medicament for the treatment or prophylaxis of Salmonella (Salmonella) and/or Escherichia coli (Escherichia coli) infections;
use of salmonella phage SAP56 in the manufacture of a medicament for the removal of salmonella and/or escherichia coli in blood. Compared with the prior art, the invention has the following advantages and effects:
the existing salmonella phage is mostly used for the food field or the disinfection of the breeding environment, but is not reported to be used for controlling systemic infection caused by salmonella and escherichia coli in livestock and poultry breeding. The bacteriophage SAP56 can crack salmonella and escherichia coli simultaneously, can enter blood through oral administration, can reach focus to prevent systemic infection caused by salmonella and escherichia coli, and provides a biological prevention and control measure which is safe, free of toxic and side effects and good in effect for mixed infection of salmonella and escherichia coli.
Drawings
FIG. 1 is a schematic of a Salmonella phage SAP56 phage panel.
Detailed Description
The invention is further illustrated below in connection with examples, but the scope of the invention as claimed is not limited to the examples. The technical scheme of the invention is conventional technology unless specified; the reagents or materials, unless otherwise specified, are commercially available.
Example 1:
salmonella phage SAP56 isolation and purification
Collecting 10 parts of water sample near some chicken farm in Hubei province, centrifuging at 30mL of each part at 6000rpm for 10min, collecting 15mL of supernatant, filtering, sterilizing, collecting 5mL of filtrate, 5mL of 2×TSB liquid medium and 1mL of salmonella bacterial liquid (10) 8 cfu/mL) allMix well, incubate at 180rpm overnight at 37℃and enrich for phages. Centrifuging the sample enrichment solution at 6000rpm for 10min, collecting supernatant, and sterilizing with microporous membrane of 0.22 μm to obtain phage enrichment solution. Mixing 100uL of filtrate with 300uL of salmonella bacteria liquid of host, adding 7mL of TSB semi-solid agar medium cooled to 50 ℃, spreading on a coagulated TSA flat plate immediately after mixing, culturing for 6-8h after agar is coagulated at 37 ℃ in an inverted mode, and observing plaque growth condition. On a double-layer plate for forming plaque, picking large transparent plaque with a sterile gun head, shaking and desorbing in 1mL SM solution, sterilizing with a microporous filter membrane of 0.22 μm to obtain phage filtrate, inoculating the phage filtrate into 5mL TSB liquid culture medium, adding 100uL of corresponding host salmonella bacteria liquid, mixing, culturing at 37 ℃ at 180rpm overnight, centrifuging at 6000rpm for 10min, filtering the supernatant with a bacterial filter membrane, and observing plaque morphology by adopting a double-layer plate method. Repeating the operation for 3-5 times to obtain phage with consistent shape and size.
Salmonella phage SAP56 was isolated from a sample of chicken house wastewater from North lake, as shown in FIG. 1, and the phage produced a single circular plaque on the Salmonella lawn, with a clear center and a diameter of 3mm. The phage was sent to China center for type culture Collection, named after classification, at day 08 and 17 of 2023: salmonella phage SAP56, address: university of martial arts, deposit number: cctccc NO: m20231500.
Example 2:
determination of optimal fermentation conditions for Salmonella phage SAP56
A single colony of the host salmonella ATCC14028 is picked, inoculated into a test tube containing 3mL of TSB culture medium, and shake-cultured for 12 hours at 180rpm in a shaking table at 37 ℃ to obtain a host bacterium suspension. The bacterial suspension was transferred to l0mL TSB medium at a ratio of 1:100, and the culture was performed by transferring a plurality of tubes and shaking at 37℃and 180 rpm. One tube per hour was removed to determine OD 600 Phage SAP56 of known titer was added and incubated for 8h with shaking at 180rpm in a shaker at 37 ℃. After the cultivation is finished, 10000g of culture solution is taken and centrifuged for 0min, the supernatant is collected, filtered and sterilized, and the phage titer is measured. The groups were averaged in duplicate tube cultures to produce the highest phageThe ratio of the potency is the optimal fermentation condition. Experiments were repeated 3 times.
The results are shown in Table 1, and the OD of the host bacterium 600 0.158, phage inoculation final concentration of 2.3X10 6 At PFU/mL, phage SAP56 titers were highest, up to 5.7X10 11 PFU/mL. The phage can therefore be used for large-scale production.
TABLE 1 determination of the optimal fermentation conditions for phages
Example 3:
PH sensitivity test of Salmonella phage SAP56
Taking sterile bacteria bottle, adding 9mL of TSB culture medium with different pH (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), placing the bacteria bottle in constant temperature water bath at 25deg.C, and adding titer of 5.3x10 after temperature balance 10 PFU/mL phage SAP56 pure culture lmL was allowed to stand at 25℃for 4h. Samples are taken at 1h, 2h and 4h respectively for proper dilution, phage titer is determined by a double-layer plate method, average value is obtained by double-tube culture at each point, and the experiment is repeated for 3 times.
As shown in Table 2, salmonella phage SAP56 was treated for 2 hours at a pH in the range of 3-11 with no significant change in potency, and after treatment for 4 hours at a pH of 4-10 with no change in potency.
TABLE 2 pH stability of Salmonella phage SAP56
Note that: ND represents not measured.
Example 4:
temperature sensitivity test of Salmonella phage SAP56
Taking a plurality of sterile 50mL centrifuge tubes, adding 9mL TSB into each centrifuge tube, placing into a constant-temperature water bath with corresponding temperature, and respectively adding the centrifuge tubes with the titer of 5.7x10 after temperature balance 11 PFU/mL phage SAP56 pure culture solution 1mL, at 35 ℃, 45 ℃, 55 ℃, 65 ℃, 75 ℃, 85 ℃ temperature in 1h, 12h, 24h, 48h, 72h. After the action time is over, the sample tube is taken out and immediately placed in an ice bath for cooling, and phage titer is measured by adopting a double-layer flat plate method after proper dilution. Each point was averaged in duplicate tube cultures and the experiment was repeated 3 times.
As shown in Table 3, the Salmonella phage SAP56 in the experimental group is easy to survive at a temperature below 65 ℃, has higher potency after water bath at 65 ℃ for 1h, has good tolerance at 35-55 ℃, and can be suitable for transportation and storage in summer after being prepared into products.
TABLE 3 temperature sensitivity of Salmonella phage SAP56
Note that: ND represents not measured.
Example 5:
salmonella phage SAP56 host range test
Taking the potency to be about 1.0x10 8 PFU/mL salmonella phage SAP56 and GSMP22333 (patent number ZL 202110735119.3) were used to determine phage host range by spot method, respectively.
The salmonella strains containing 11 serotypes and the escherichia coli strains containing 12 serotypes separated from clinic 26 strains are selected, inoculated into a centrifuge tube containing 3mL of TSB respectively, and cultured for 8 hours at 37 ℃ at 180rpm, so that bacterial suspensions of the strains are prepared. 300uL of bacterial suspension is mixed with TSB semi-solid culture medium and spread on a prepared TSA plate, and 5uL of phage culture liquid is dripped on the plate. After natural air drying, culturing for 5-8h at 37 ℃ and observing the result. The test was repeated three times.
As a result, as shown in Table 4, phage SAP56 can lyse 21 strains of Salmonella containing 11 sera at a lysis rate of 80.8% and 17 strains of E.coli containing 10 serotypes at a lysis rate of 77.3%; while the phage GSMP22333 had a Salmonella lysis rate of 65.4% and E.coli lysis rate of 22.7%. And the splitting effect of the SAP56 is better than that of GSMP22333, which shows that the phage SAP56 has potential for preparing biological agents for preventing and controlling salmonella and escherichia coli mixed infection.
TABLE 4 Salmonella phage SAP56 host range
Note that: "-" means not cleaved; "+" indicates slight cracking and cracked spot blurring; "++" indicates cracking, and cracking spots are clearer; "+". ++'s indicating the cleavage of the sample, the cleavage spots are very clear.
Example 6:
phage SAP56 fermentation preparation:
a single colony of the host salmonella ATCC14028 is picked, inoculated into a test tube containing 3mL of TSB culture solution, and shake-cultured for 12 hours at 180rpm in a shaking table at 37 ℃ to obtain a host bacterium suspension. The bacterial suspension was transferred to 500mL of TSB medium at a ratio of 1:100, cultured with shaking at 37℃and 180rpm to the log phase and the bacterial suspension concentration was determined. The system prepared by fermentation is 6L, and the fermentation medium is TSB medium. Inoculating by flame inoculation, and inoculating under optimal infection conditions (host bacteria OD 600 0.158, phage SAP56 concentration of 4.5x10 6 PFU/mL) was inoculated with phage SAP56 and Salmonella ATCC14028 into the fermentation medium. Sterile air is introduced in the fermentation process, and 3 per mill of defoamer is added, so that the fermentation preparation time is 10 hours. 20mL of the fermentation broth was taken from the sampling port every 2 hours from the start of fermentation in a sterile container, centrifuged at 5000rpm for 10min, and the supernatant was sterilized by passing through a microporous filter membrane of 0.22 μm to obtain a phage-containing filtrate, and the titer thereof was measured, in the same manner as described in example 2.
After fermentation, taking out the whole mixed liquid of phage and host bacteria from the sampling port, inoculating into a sterile container, centrifuging at 6000rpm for 15min, taking supernatant, filtering into a sterile filter device by a vacuum pump, obtaining phage fermentation liquid, and preserving at 4 ℃.
As can be seen from Table 5, phage SAP56 aged up to 6.6x10 at 6h of fermentation 11 PFU/mL. Thereafter, the phage titer is not substantially changed, and thus, large-scale industrial production of phage by fermentation is feasible.
TABLE 5 fermentation kinetics of Salmonella phage SAP56
Example 7:
application of phage SAP56 in prevention and treatment of mixed infection of salmonella and escherichia coli
40 blood phage negative egg chicks were selected and randomly divided into 4 groups (SAP 56 group, GSMP22333 group, challenge group and blank group), 10 per group, and 10 SAP56 group after 3 days of pre-feeding 7 cfu/kg salmonella and 10 7 cfu/kg of escherichia coli, and 10 is drenched after 12 hours 8 pfu/kg phage SAP56; GSMP22333 group administration 10 7 cfu/kg salmonella and 10 7 cfu/kg of escherichia coli, and 10 is drenched after 12 hours 8 pfu/kg phage GSMP22333; administration of the offensive drug to the group 10 7 cfu/kg salmonella and 10 7 cfu/kg of E.coli; the blank group was given an equal amount of PBS buffer. And continuously observing for 5 days, recording death conditions of the chicks, dissecting all surviving chicks after the experiment is finished, and recording phage content in serum and liver bacteria-carrying conditions.
As shown in the results of Table 6, phage were detected in blood of SAP56 group chicks at a level of 3.1X10 5 The phage content in the blood of PFU/mL and GSMP22333 group is 710PFU/mL, and no phage is detected in the blood of the other 2 groups of chickens, which indicates that phage SAP56 and GSMP22333 can enter the blood through oral administration, but GSMP22333 does not enter the blood as well as phage SAP56.
The results in Table 7 show that the phage SAP56 can lyse salmonella and escherichia coli, has good control effect on mixed infection of salmonella and escherichia coli, enters blood to reach the liver, and has a cleaning effect on salmonella and escherichia coli in the liver, so that the survival rate of chickens is improved. The phage GSMP22333 has no effect of splitting escherichia coli and has poor blood inflow effect, so that the prevention and control effect on the mixed infection of salmonella and escherichia coli of the chicks are inferior to those of phage SAP56.
TABLE 6 phage content in chick serum
TABLE 7 Effect of phage SAP56 on application in Salmonella and E.coli Mixed infection in chickens
| Grouping | SAP56 group | GSMP22333 group | Toxin eliminating group | Blank group |
| Survival rate | 100％ | 50％ | 10％ | 100％ |
| Positive rate of salmonella hepatica | 0 | 60％ | 100％ | 0 |
| Liver E.coli Positive rate | 0 | 100％ | 100％ | 0 |
The results show that the bacteriophage SAP56 has strong environmental adaptability and can be used for industrial large-scale fermentation production, has the characteristics of simultaneously lysing salmonella and escherichia coli and orally taking blood, and can be used for preparing biological agents for preventing and controlling systemic infection caused by the mixing of the salmonella and the escherichia coli.
Claims (3)
1. An isolated Salmonella phage SAP56, said phage having a accession number: cctccc NO: m20231500.
2. The salmonella bacteriophage SAP56 of claim 1 for use in the preparation of a medicament for the treatment or prophylaxis of salmonellaSalmonella) And/or Escherichia coliEscherichia coli) Use of an infected drug.
3. Use of the salmonella bacteriophage SAP56 of claim 1, for the manufacture of a medicament for the removal of salmonella and/or escherichia coli in blood.
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| CN118147090A (en) * | 2024-05-10 | 2024-06-07 | 烟台金海药业有限公司 | Phage for simultaneously lysing multiple strains of escherichia coli and salmonella and application thereof |
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| CN118147090A (en) * | 2024-05-10 | 2024-06-07 | 烟台金海药业有限公司 | Phage for simultaneously lysing multiple strains of escherichia coli and salmonella and application thereof |
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