CN117487767B - Method for improving phage outbreak by using low-concentration antibiotics and application - Google Patents
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Abstract
The invention belongs to the technical field of microorganisms, and discloses a method for improving phage outbreak by using low-concentration antibiotics and application thereof. After the method is used for inoculating host bacteria into a sterilized culture medium, antibiotics are added, salmonella phage is added after the culture temperature and dissolved oxygen are increased, aerobic culture is carried out, so that the time for proliferation of the salmonella phage in the host bacteria is prolonged, and the outbreak amount of the salmonella phage and the titer of a proliferation liquid are increased. According to the invention, proper antibiotics are added in the phage multiplication process, so that the multiplication time of phage in a host is prolonged, the explosion quantity of phage and the titer of multiplication liquid are increased, and the quality of a product is improved. By optimizing the salmonella phage production process, the titer of phage multiplication liquid can be greatly improved, the production cost is reduced, the use effect of the product is improved, the use cost is reduced, and the economic benefit is improved.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a method for improving phage outbreak by using low-concentration antibiotics and application thereof.
Background
Salmonella is a common food-borne pathogen that is widely present in nature and has a range of damage to humans from mild intestinal inflammation to fatal systemic infection. Antibiotics are mostly used for clinically treating and preventing infection caused by salmonella, and the excessive use of the antibiotics causes the situation that the salmonella has increasingly serious drug resistance.
Phage is a virus that attacks bacteria, present in a variety of environments, such as soil, sea water, animal intestines, and is considered the most abundant organism on earth. Phages have strict host specificity and must be parasitic in living bacteria, which depends on the molecular structure and complementarity of the phage's adsorbing organ with the host bacteria surface receptor.
Phage is a bacterial virus that is widely distributed in nature and can specifically lyse bacteria. Phage therapy has the advantages that: strong specificity, self-replication and proliferation, safety, wide sources and the like, and is a biological control technology which can replace antibiotics and has very good prospect. Phage therapy has the advantages of strong specificity, self proliferation, safety, wide sources and the like, and is a biological control technology with great prospect.
In order to improve the titer of the phage multiplication liquid, the conventional method has limited titer improvement of the multiplication liquid by optimizing the components of a culture medium, adjusting the access time of phage, improving the rotating speed and ventilation of a fermentation tank and the like, and cannot greatly improve the titer of the multiplication liquid.
Disclosure of Invention
In order to overcome the problems in the related art, the disclosed embodiments of the present invention provide a method for increasing the amount of phage display by using low concentration antibiotics and application thereof.
The technical scheme is as follows: the method for improving the explosion quantity of the phage by using the low-concentration antibiotics comprises the steps of inoculating host bacteria into a sterilized culture medium, adding the antibiotics, improving the culture temperature and dissolved oxygen, adding salmonella phage, and performing aerobic culture to prolong the proliferation time of the salmonella phage in the host bacteria and increase the explosion quantity of the salmonella phage and the titer of a proliferation liquid.
Further, in the presence of phage, a 96-well plate was used to determine that 3% of the ratio of the host bacteria, salmonella S6, was added to the sterilized medium.
Further, lincomycin antibiotic was added at a concentration of 1/8 MIC.
Further, after the addition of the antibiotic, the culture was carried out at 37℃with a aeration rate of 0.8vvm at 200rpm for 3 hours.
Further, after raising the culture temperature and dissolved oxygen, the salmonella phage was inoculated at a ratio of 3%.
The salmonella phage is named as RDP-SA-17118, the preservation number is 18198, the preservation unit is China general microbiological culture Collection center (China Committee for culture Collection of microorganisms), the preservation address is North Star Xiyu No.1, 3 of the Korean area of Beijing, the preservation date is 2019, the month is 07, and the classification is named as: salmonella phage ESCHERICHIA COLI survived.
Further, the temperature of the fermentation tank is adjusted to 38 ℃, the aeration rate is 1vvm, the rotating speed is 300rpm, and after the fermentation is carried out for 7 to 8 hours, the proliferation is finished after the pH and the dissolved oxygen are stabilized.
Further, the medium comprises: 2.0% yeast extract, 0.5% yeast peptone, 0.5% glycerol, 0.9% sodium chloride, 10ppm calcium chloride, 10ppm magnesium chloride, and the rest is supplemented with water.
Further, the medium was sterilized at pH 7.4.+ -. 0.2, 121℃for 30 min.
Further, the screening method of lincomycin antibiotics added with 1/8MIC concentration comprises the following steps:
Inoculating host bacteria with 3% of inoculum size, selecting salmonella from the host bacteria, culturing at 37 ℃ and 200rpm for 3 hours, and inoculating bacteriophage with 3% of inoculum size; adding antibiotics with different concentrations, wherein the final concentration of the antibiotics is 1/2, 1/4, 1/8 and 1/16 of the minimum sensitization concentration respectively, and measuring the burst size and proliferation liquid titer after culturing;
Average lysis = phage titer at end of outbreak/host concentration at initial stage of infection;
average burst size = phage titer at end of burst/host concentration at initial stage of infection.
Another object of the invention is the use of said method for increasing the burst size of phages with low concentration of antibiotics for the preparation of a medicament for the treatment and prevention of intestinal inflammation.
By combining all the technical schemes, the invention has the advantages and positive effects that: according to the invention, proper antibiotics are added in the phage multiplication process, so that the multiplication time of phage in a host is prolonged, the explosion quantity of phage and the titer of multiplication liquid are increased, and the quality of a product is improved. By optimizing the salmonella phage production process, the titer of phage multiplication liquid can be greatly improved, the production cost is reduced, the use effect of the product is improved, the use cost is reduced, and the economic benefit is improved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the disclosure and together with the description, serve to explain the principles of the disclosure;
FIG. 1 is a flow chart of a method for increasing phage outbreak using low concentration antibiotics according to an embodiment of the present invention.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit or scope of the invention, which is therefore not limited to the specific embodiments disclosed below.
The method for improving phage outbreak by using low-concentration antibiotics provided by the embodiment of the invention has the innovation points that: through low-concentration antibiotics, the explosion quantity of phage is effectively increased, the titer of phage proliferation liquid is improved, the quality and quality of products are improved, and the production cost is reduced.
After host bacteria are inoculated into the sterilized culture medium, antibiotics are added, salmonella phage is added after the culture temperature and dissolved oxygen are increased, and aerobic culture is carried out, so that the proliferation time of the salmonella phage in the host bacteria is prolonged, and the outbreak amount of the salmonella phage and the titer of a proliferation liquid are increased.
Example 1, the method for increasing phage outbreaks using low concentration antibiotics provided by the example of the invention comprises:
in the phage multiplication process, proper antibiotics are added, and the time for phage multiplication in a host body is prolonged by methods of raising the culture temperature, dissolving oxygen and the like, so that the explosion quantity of phage and the titer of multiplication liquid are improved.
As shown in fig. 1, the method specifically includes:
s1, determining MIC of antibiotics in the presence of phage by using a 96-well plate, and adding 1/8MIC concentration of antibiotics (filtering and sterilizing by using a 0.22 μm membrane) into a sterilized culture medium;
S2, after 1/8MIC concentration of antibiotics is added, the culture is aerobically cultured for 3 hours at 37 ℃;
s3, after aerobic culture for 3 hours, 3% of phage is added, and further aerobic culture is carried out for 6-7 hours, and proliferation is finished.
In order to achieve the aim of improving the burst quantity and the titer of the proliferation liquid, the culture conditions such as initial pH, culture temperature, ventilation quantity and the like are further optimized by optimizing the types, the addition quantity and the addition time of the antibiotics.
Example 2 selection of different antibiotics:
Minimum drug sensitivity concentration determination when only antibiotics are present: a 96-well plate method was used.
Materials: antibiotics (apramycin, doxycycline, neomycin, aj Mo Xinlin, lincomycin, colistin sulfate, kanamycin, aureomycin), 100-fold diluted host bacteria (S6), and salmonella phage (RDP-SA-17118), LB liquid medium at a concentration of 1.024%.
The salmonella phage is named as RDP-SA-17118, the preservation number is 18198, the preservation unit is China general microbiological culture Collection center (China Committee for culture Collection of microorganisms), the preservation address is No. 3 of the West Song No.1 of North Star in the Korean area of Beijing, the preservation date is 2019, the month is 07, and the classification is named as: salmonella phage ESCHERICHIA COLI survived.
The method comprises the following steps: 100. Mu.L of LB liquid medium was added to each 96 wells, 100. Mu.L of antibiotics were added sequentially to the first row of wells, diluted sequentially from left to right with a row gun, blown 10 times with a row gun each time until column 11, the excess 100. Mu.L of liquid was discarded, and then 100. Mu.L of diluted Salmonella (S6) was added to each well, column 12 served as a positive control. The prepared 96-well plate was placed in an incubator at 37℃and the results were observed after 16-24 hours, and the results are shown in Table 1.
TABLE 1
Minimum drug sensitivity concentration determination in the presence of phage: a 96-well plate method was used.
Salmonella phage and antibiotics have good synergistic effect, and if the concentration of the antibiotics is too high, the growth of host bacteria can be inhibited, so that the proliferation of phage is affected. For this purpose, the minimum susceptibility concentration of the antibiotic when phage is present is determined.
The method comprises the following steps: 2 96-well plates were placed side by side, 100. Mu.L of LB liquid medium was added in each 96-well, 100. Mu.L of antibiotics were added in the first row of wells in sequence, diluted sequentially from left to right with a row gun, 10 strokes were performed with a row gun each time until column 23 was reached, excess 100. Mu.L of liquid was discarded, then 100. Mu.L of host bacteria (Salmonella (S6)) and Salmonella phage (RDP-SA-17118) diluted 100-fold were added per well, column 24 was used as a positive control, and 100. Mu.L of Salmonella (S6) diluted 100-fold was added. The prepared 96-well plate was placed in an incubator at 37℃and the observation results after 16-24 hours are shown in Table 2.
TABLE 2
| Sequence number | Antibiotic + phage | Minimum dilution factor 2 n |
| 1 | Apramycin + phage | 12 |
| 2 | Doxycycline + phage | 14 |
| 3 | Neomycin + phage | 13 |
| 4 | A Mo Xinlin + phage | 8 |
| 5 | Lincomycin + phage | 11 |
| 6 | Colistin sulfate + phage | 14 |
| 7 | Kanamycin+ phage | 12 |
| 8 | Aureomycin + phage | 11 |
When phage exists, the minimum drug sensitivity of antibiotics is greatly reduced, which indicates that the antibiotics and the antibiotics have synergistic effect.
When phage and antibiotics are present at the same time, the concentration of antibiotics needs to be optimized in order to avoid too high an antibiotic concentration and to inhibit the growth of host bacteria.
Example 2: selection of antibiotic concentration:
salmonella phage and antibiotics have good synergistic effect, and if the concentration of the antibiotics is too high, the growth of host bacteria can be inhibited, so that the proliferation of phage is affected.
Inoculating 3% of the inoculum size into a host, culturing at 37 ℃ at 200rpm for 3 hours, inoculating 3% of the inoculum size into phage, adding antibiotics with different concentrations, wherein the final concentration of the antibiotics is 1/2, 1/4, 1/8 and 1/16 of the minimum sensitization concentration respectively, and measuring the burst size and proliferation liquid titer after culturing for 8 hours.
Average amount of cleavage: average lysis = phage titer at end of outbreak/host concentration at the beginning of infection.
Burst size: average burst size = phage titer at end of burst/host concentration at initial stage of infection.
TABLE 3 Table 3
TABLE 4 Table 4
As can be seen from tables 3 and 4, in the phage multiplication process, the addition of antibiotics with concentration lower than drug sensitivity can effectively increase the burst size of phage, and can greatly increase the titer of multiplication liquid, and lincomycin, neomycin, apramycin, kanamycin, doxycycline, aureomycin, colistin sulfate and amoxicillin, among 8 antibiotics (Chinese drugs), the lincomycin has the best effect.
Example 4: access timing of antibiotics.
And (5) verifying lincomycin with the best effect, and checking the access time of antibiotics.
Host bacteria (salmonella (S6)) were inoculated at 3% inoculum size, and then antibiotics were added at various times. Culturing at 200-300rpm at 37-38deg.C for 3-3.5 hr, and inoculating phage at 3-5% inoculum size. Lincomycin at 1/8MIC (final concentration) was added at various times after host access. And (3) proliferation is carried out for 7-8 hours, and after a large amount of floccules appear, the proliferation is finished, and the titers of the floccules are respectively measured.
After the host is cultured for 3 hours, phage is inoculated, and lincomycin is added within 0-30min after phage inoculation, so that the effect is optimal, the burst size is 385, and the proliferation liquid titer is maximum (1.53E12 CFU/mL).
TABLE 5
| Sequence number | Time (min) | Burst size | Valence (CFU/mL) | Remarks |
| 1 | 0 | 312 | 9.62E+11 | |
| 2 | 30 | 326 | 1.16E+12 | |
| 3 | 60 | 239 | 1.21E+12 | |
| 4 | 90 | 348 | 1.29E+12 | |
| 5 | 120 | 356 | 1.36E+12 | |
| 6 | 150 | 361 | 1.42E+12 | |
| 7 | 180 | 376 | 1.49E+12 | 3% Phage access |
| 8 | 210 | 385 | 1.53E+12 | |
| 9 | 240 | 362 | 1.33E+12 |
Example 5: a salmonella phage proliferation process.
The above process was verified using a 10L fermenter and further optimized for fermentation parameters.
Culture medium: 2.0% yeast extract, 0.5% yeast peptone, 0.5% glycerol, 0.9% sodium chloride, 10ppm calcium chloride, 10ppm magnesium chloride, pH 7.4+ -0.2,121 deg.C, and sterilizing for 30 min.
After sterilization, salmonella is inoculated in a proportion of 3 percent (S6), 1/8MIC lincomycin is added, the culture temperature is 37-38 ℃, the ventilation rate is 0.8-0.9vvm, the rotation speed is 200-300rpm, bacteriophage RP-SA-17118 is inoculated in a proportion of 3-5 percent after 3-3.5 hours of culture, the temperature of a fermentation tank is adjusted to 38 ℃, the ventilation rate is 1-1.1vvm, the rotation speed is 300-350rpm, and after 7-8 hours of proliferation, the proliferation is finished after the pH and dissolved oxygen are stable.
In the foregoing embodiments, the descriptions of the embodiments are emphasized, and in part, not described or illustrated in any particular embodiment, reference is made to the related descriptions of other embodiments.
While the invention has been described with respect to what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but on the contrary, is intended to cover various modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Claims (4)
1. A method for increasing phage outbreak using a low concentration of an antibiotic, the method comprising: inoculating 3% of host bacteria in the sterilized culture medium, culturing the host bacteria for 3 hours at 37 ℃ and 200rpm, and inoculating bacteriophage at an inoculum size of 3%; adding lincomycin antibiotics at a concentration of 1/8 MIC; measuring burst size and proliferation liquid titer after culturing;
The salmonella phage is named as RDP-SA-17118, the preservation number is 18198, the preservation unit is China general microbiological culture Collection center (China Committee for culture Collection of microorganisms), the preservation address is No. 3 of the West Song No.1 of North Star in the Korean area of Beijing, the preservation date is 2019, the month is 07, and the classification is named as: salmonella phage ESCHERICHIA COLI survived.
2. The method for increasing phage outbreak using low concentration antibiotics according to claim 1, wherein the medium comprises: 2.0% yeast extract, 0.5% yeast peptone, 0.5% glycerol, 0.9% sodium chloride, 10ppm calcium chloride, 10ppm magnesium chloride, and the rest is supplemented with water.
3. The method for increasing phage display explosive amount by using low concentration antibiotics according to claim 2, wherein the medium is sterilized at ph7.4±0.2, 121 ℃ for 30min.
4. The method of increasing phage outbreak using low concentration antibiotics according to claim 1, wherein mean lytic amount = phage titer at end of outbreak/host concentration at initial stage of infection;
average burst size = phage titer at end of burst/host concentration at initial stage of infection.
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| CN117050954A (en) * | 2023-07-21 | 2023-11-14 | 青岛农业大学 | Broad-spectrum salmonella phage vB-SenS-S1 and composition containing phage |
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| JP5292278B2 (en) * | 2006-04-04 | 2013-09-18 | サントル ナショナル ドゥ ラ ルシェルシュ スィヤンティフィック（セーエヌエルエス） | Method for producing bacteriophage composition and method in the field of phage therapy |
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| KR101909831B1 (en) * | 2017-08-28 | 2018-10-18 | 한국외국어대학교 연구산학협력단 | Composition and method of increasing production of bacteriophage by use of Reactive oxygen species |
| CN112063594B (en) * | 2020-09-24 | 2022-02-11 | 瑞科盟(青岛)生物工程有限公司 | High-temperature-resistant salmonella bacteriophage RDP-SA-18056 and preparation process of microcapsules thereof |
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| CN113583972B (en) * | 2021-08-05 | 2022-07-22 | 瑞科盟(青岛)生物工程有限公司 | Escherichia coli bacteriophage capable of reducing antibiotic resistance and application thereof |
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| CN114015661B (en) * | 2021-12-14 | 2024-06-25 | 青岛润达生物科技有限公司 | Culture medium and method for improving phage titer |
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| CN117050954A (en) * | 2023-07-21 | 2023-11-14 | 青岛农业大学 | Broad-spectrum salmonella phage vB-SenS-S1 and composition containing phage |
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